diff --git a/experimental/CHANGELOG.md b/experimental/CHANGELOG.md
index 688384d220..f9ead87184 100644
--- a/experimental/CHANGELOG.md
+++ b/experimental/CHANGELOG.md
@@ -8,6 +8,7 @@ All notable changes to experimental packages in this project will be documented
 
 ### :rocket: (Enhancement)
 
+* feat(instrumentation): implement `require-in-the-middle` singleton [#3161](https://github.com/open-telemetry/opentelemetry-js/pull/3161) @mhassan1
 * feat(sdk-node): configure trace exporter with environment variables [#3143](https://github.com/open-telemetry/opentelemetry-js/pull/3143) @svetlanabrennan
 * feat: enable tree shaking [#3329](https://github.com/open-telemetry/opentelemetry-js/pull/3329) @pkanal
 * feat(prometheus): serialize resource as target_info gauge [#3300](https://github.com/open-telemetry/opentelemetry-js/pull/3300) @pichlermarc
diff --git a/experimental/packages/opentelemetry-instrumentation/src/platform/node/ModuleNameTrie.ts b/experimental/packages/opentelemetry-instrumentation/src/platform/node/ModuleNameTrie.ts
new file mode 100644
index 0000000000..3230fea99c
--- /dev/null
+++ b/experimental/packages/opentelemetry-instrumentation/src/platform/node/ModuleNameTrie.ts
@@ -0,0 +1,86 @@
+/*
+ * Copyright The OpenTelemetry Authors
+ *
+ * Licensed under the Apache License, Version 2.0 (the "License");
+ * you may not use this file except in compliance with the License.
+ * You may obtain a copy of the License at
+ *
+ *      https://www.apache.org/licenses/LICENSE-2.0
+ *
+ * Unless required by applicable law or agreed to in writing, software
+ * distributed under the License is distributed on an "AS IS" BASIS,
+ * WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied.
+ * See the License for the specific language governing permissions and
+ * limitations under the License.
+ */
+
+import type { Hooked } from './RequireInTheMiddleSingleton';
+
+export const ModuleNameSeparator = '/';
+
+/**
+ * Node in a `ModuleNameTrie`
+ */
+class ModuleNameTrieNode {
+  hooks: Array<{ hook: Hooked, insertedId: number }> = [];
+  children: Map<string, ModuleNameTrieNode> = new Map();
+}
+
+/**
+ * Trie containing nodes that represent a part of a module name (i.e. the parts separated by forward slash)
+ */
+export class ModuleNameTrie {
+  private _trie: ModuleNameTrieNode = new ModuleNameTrieNode();
+  private _counter: number = 0;
+
+  /**
+   * Insert a module hook into the trie
+   *
+   * @param {Hooked} hook Hook
+   */
+  insert(hook: Hooked) {
+    let trieNode = this._trie;
+
+    for (const moduleNamePart of hook.moduleName.split(ModuleNameSeparator)) {
+      let nextNode = trieNode.children.get(moduleNamePart);
+      if (!nextNode) {
+        nextNode = new ModuleNameTrieNode();
+        trieNode.children.set(moduleNamePart, nextNode);
+      }
+      trieNode = nextNode;
+    }
+    trieNode.hooks.push({ hook, insertedId: this._counter++ });
+  }
+
+  /**
+   * Search for matching hooks in the trie
+   *
+   * @param {string} moduleName Module name
+   * @param {boolean} maintainInsertionOrder Whether to return the results in insertion order
+   * @returns {Hooked[]} Matching hooks
+   */
+  search(moduleName: string, { maintainInsertionOrder }: { maintainInsertionOrder?: boolean } = {}): Hooked[] {
+    let trieNode = this._trie;
+    const results: ModuleNameTrieNode['hooks'] = [];
+
+    for (const moduleNamePart of moduleName.split(ModuleNameSeparator)) {
+      const nextNode = trieNode.children.get(moduleNamePart);
+      if (!nextNode) {
+        break;
+      }
+      results.push(...nextNode.hooks);
+      trieNode = nextNode;
+    }
+
+    if (results.length === 0) {
+      return [];
+    }
+    if (results.length === 1) {
+      return [results[0].hook];
+    }
+    if (maintainInsertionOrder) {
+      results.sort((a, b) => a.insertedId - b.insertedId);
+    }
+    return results.map(({ hook }) => hook);
+  }
+}
diff --git a/experimental/packages/opentelemetry-instrumentation/src/platform/node/RequireInTheMiddleSingleton.ts b/experimental/packages/opentelemetry-instrumentation/src/platform/node/RequireInTheMiddleSingleton.ts
new file mode 100644
index 0000000000..812db52b68
--- /dev/null
+++ b/experimental/packages/opentelemetry-instrumentation/src/platform/node/RequireInTheMiddleSingleton.ts
@@ -0,0 +1,111 @@
+/*
+ * Copyright The OpenTelemetry Authors
+ *
+ * Licensed under the Apache License, Version 2.0 (the "License");
+ * you may not use this file except in compliance with the License.
+ * You may obtain a copy of the License at
+ *
+ *      https://www.apache.org/licenses/LICENSE-2.0
+ *
+ * Unless required by applicable law or agreed to in writing, software
+ * distributed under the License is distributed on an "AS IS" BASIS,
+ * WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied.
+ * See the License for the specific language governing permissions and
+ * limitations under the License.
+ */
+
+import * as RequireInTheMiddle from 'require-in-the-middle';
+import * as path from 'path';
+import { ModuleNameTrie, ModuleNameSeparator } from './ModuleNameTrie';
+
+export type Hooked = {
+  moduleName: string
+  onRequire: RequireInTheMiddle.OnRequireFn
+};
+
+/**
+ * Whether Mocha is running in this process
+ * Inspired by https://github.com/AndreasPizsa/detect-mocha
+ *
+ * @type {boolean}
+ */
+const isMocha = ['afterEach','after','beforeEach','before','describe','it'].every(fn => {
+  // @ts-expect-error TS7053: Element implicitly has an 'any' type
+  return typeof global[fn] === 'function';
+});
+
+/**
+ * Singleton class for `require-in-the-middle`
+ * Allows instrumentation plugins to patch modules with only a single `require` patch
+ * WARNING: Because this class will create its own `require-in-the-middle` (RITM) instance,
+ * we should minimize the number of new instances of this class.
+ * Multiple instances of `@opentelemetry/instrumentation` (e.g. multiple versions) in a single process
+ * will result in multiple instances of RITM, which will have an impact
+ * on the performance of instrumentation hooks being applied.
+ */
+export class RequireInTheMiddleSingleton {
+  private _moduleNameTrie: ModuleNameTrie = new ModuleNameTrie();
+  private static _instance?: RequireInTheMiddleSingleton;
+
+  private constructor() {
+    this._initialize();
+  }
+
+  private _initialize() {
+    RequireInTheMiddle(
+      // Intercept all `require` calls; we will filter the matching ones below
+      null,
+      { internals: true },
+      (exports, name, basedir) => {
+        // For internal files on Windows, `name` will use backslash as the path separator
+        const normalizedModuleName = normalizePathSeparators(name);
+
+        const matches = this._moduleNameTrie.search(normalizedModuleName, { maintainInsertionOrder: true });
+
+        for (const { onRequire } of matches) {
+          exports = onRequire(exports, name, basedir);
+        }
+
+        return exports;
+      }
+    );
+  }
+
+  /**
+   * Register a hook with `require-in-the-middle`
+   *
+   * @param {string} moduleName Module name
+   * @param {RequireInTheMiddle.OnRequireFn} onRequire Hook function
+   * @returns {Hooked} Registered hook
+   */
+  register(moduleName: string, onRequire: RequireInTheMiddle.OnRequireFn): Hooked {
+    const hooked = { moduleName, onRequire };
+    this._moduleNameTrie.insert(hooked);
+    return hooked;
+  }
+
+  /**
+   * Get the `RequireInTheMiddleSingleton` singleton
+   *
+   * @returns {RequireInTheMiddleSingleton} Singleton of `RequireInTheMiddleSingleton`
+   */
+  static getInstance(): RequireInTheMiddleSingleton {
+    // Mocha runs all test suites in the same process
+    // This prevents test suites from sharing a singleton
+    if (isMocha) return new RequireInTheMiddleSingleton();
+
+    return this._instance = this._instance ?? new RequireInTheMiddleSingleton();
+  }
+}
+
+/**
+ * Normalize the path separators to forward slash in a module name or path
+ *
+ * @param {string} moduleNameOrPath Module name or path
+ * @returns {string} Normalized module name or path
+ */
+function normalizePathSeparators(moduleNameOrPath: string): string {
+  return path.sep !== ModuleNameSeparator
+    ? moduleNameOrPath.split(path.sep).join(ModuleNameSeparator)
+    : moduleNameOrPath;
+}
diff --git a/experimental/packages/opentelemetry-instrumentation/src/platform/node/instrumentation.ts b/experimental/packages/opentelemetry-instrumentation/src/platform/node/instrumentation.ts
index 5bc0921778..d80985431c 100644
--- a/experimental/packages/opentelemetry-instrumentation/src/platform/node/instrumentation.ts
+++ b/experimental/packages/opentelemetry-instrumentation/src/platform/node/instrumentation.ts
@@ -16,9 +16,9 @@
 
 import * as types from '../../types';
 import * as path from 'path';
-import * as RequireInTheMiddle from 'require-in-the-middle';
 import { satisfies } from 'semver';
 import { InstrumentationAbstract } from '../../instrumentation';
+import { RequireInTheMiddleSingleton, Hooked } from './RequireInTheMiddleSingleton';
 import { InstrumentationModuleDefinition } from './types';
 import { diag } from '@opentelemetry/api';
 
@@ -29,7 +29,8 @@ export abstract class InstrumentationBase<T = any>
   extends InstrumentationAbstract
   implements types.Instrumentation {
   private _modules: InstrumentationModuleDefinition<T>[];
-  private _hooks: RequireInTheMiddle.Hooked[] = [];
+  private _hooks: Hooked[] = [];
+  private _requireInTheMiddleSingleton: RequireInTheMiddleSingleton = RequireInTheMiddleSingleton.getInstance();
   private _enabled = false;
 
   constructor(
@@ -160,9 +161,8 @@ export abstract class InstrumentationBase<T = any>
     this._warnOnPreloadedModules();
     for (const module of this._modules) {
       this._hooks.push(
-        RequireInTheMiddle(
-          [module.name],
-          { internals: true },
+        this._requireInTheMiddleSingleton.register(
+          module.name,
           (exports, name, baseDir) => {
             return this._onRequire<typeof exports>(
               (module as unknown) as InstrumentationModuleDefinition<
diff --git a/experimental/packages/opentelemetry-instrumentation/test/node/ModuleNameTrie.test.ts b/experimental/packages/opentelemetry-instrumentation/test/node/ModuleNameTrie.test.ts
new file mode 100644
index 0000000000..c3d72c89d7
--- /dev/null
+++ b/experimental/packages/opentelemetry-instrumentation/test/node/ModuleNameTrie.test.ts
@@ -0,0 +1,68 @@
+/*
+ * Copyright The OpenTelemetry Authors
+ *
+ * Licensed under the Apache License, Version 2.0 (the "License");
+ * you may not use this file except in compliance with the License.
+ * You may obtain a copy of the License at
+ *
+ *      https://www.apache.org/licenses/LICENSE-2.0
+ *
+ * Unless required by applicable law or agreed to in writing, software
+ * distributed under the License is distributed on an "AS IS" BASIS,
+ * WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied.
+ * See the License for the specific language governing permissions and
+ * limitations under the License.
+ */
+
+import * as assert from 'assert';
+import { Hooked } from '../../src/platform/node/RequireInTheMiddleSingleton';
+import { ModuleNameTrie } from '../../src/platform/node/ModuleNameTrie';
+
+describe('ModuleNameTrie', () => {
+  describe('search', () => {
+    const trie = new ModuleNameTrie();
+    const inserts = [
+      { moduleName: 'a', onRequire: () => {} },
+      { moduleName: 'a/b', onRequire: () => {} },
+      { moduleName: 'a', onRequire: () => {} },
+      { moduleName: 'a/c', onRequire: () => {} },
+      { moduleName: 'd', onRequire: () => {} }
+    ] as Hooked[];
+    inserts.forEach(trie.insert.bind(trie));
+
+    it('should return a list of exact matches (no results)', () => {
+      assert.deepEqual(trie.search('e'), []);
+    });
+
+    it('should return a list of exact matches (one result)', () => {
+      assert.deepEqual(trie.search('d'), [inserts[4]]);
+    });
+
+    it('should return a list of exact matches (more than one result)', () => {
+      assert.deepEqual(trie.search('a'), [
+        inserts[0],
+        inserts[2]
+      ]);
+    });
+
+    describe('maintainInsertionOrder = false', () => {
+      it('should return a list of matches in prefix order', () => {
+        assert.deepEqual(trie.search('a/b'), [
+          inserts[0],
+          inserts[2],
+          inserts[1]
+        ]);
+      });
+    });
+
+    describe('maintainInsertionOrder = true', () => {
+      it('should return a list of matches in insertion order', () => {
+        assert.deepEqual(trie.search('a/b', { maintainInsertionOrder: true }), [
+          inserts[0],
+          inserts[1],
+          inserts[2]
+        ]);
+      });
+    });
+  });
+});
diff --git a/experimental/packages/opentelemetry-instrumentation/test/node/RequireInTheMiddleSingleton.test.ts b/experimental/packages/opentelemetry-instrumentation/test/node/RequireInTheMiddleSingleton.test.ts
new file mode 100644
index 0000000000..724dced720
--- /dev/null
+++ b/experimental/packages/opentelemetry-instrumentation/test/node/RequireInTheMiddleSingleton.test.ts
@@ -0,0 +1,126 @@
+/*
+ * Copyright The OpenTelemetry Authors
+ *
+ * Licensed under the Apache License, Version 2.0 (the "License");
+ * you may not use this file except in compliance with the License.
+ * You may obtain a copy of the License at
+ *
+ *      https://www.apache.org/licenses/LICENSE-2.0
+ *
+ * Unless required by applicable law or agreed to in writing, software
+ * distributed under the License is distributed on an "AS IS" BASIS,
+ * WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied.
+ * See the License for the specific language governing permissions and
+ * limitations under the License.
+ */
+
+import * as assert from 'assert';
+import * as sinon from 'sinon';
+import * as path from 'path';
+import * as RequireInTheMiddle from 'require-in-the-middle';
+import { RequireInTheMiddleSingleton } from '../../src/platform/node/RequireInTheMiddleSingleton';
+
+const requireInTheMiddleSingleton = RequireInTheMiddleSingleton.getInstance();
+
+type AugmentedExports = {
+  __ritmOnRequires?: string[]
+};
+
+const makeOnRequiresStub = (label: string): sinon.SinonStub => sinon.stub().callsFake(((exports: AugmentedExports) => {
+  exports.__ritmOnRequires ??= [];
+  exports.__ritmOnRequires.push(label);
+  return exports;
+}) as RequireInTheMiddle.OnRequireFn);
+
+describe('RequireInTheMiddleSingleton', () => {
+  describe('register', () => {
+    const onRequireFsStub = makeOnRequiresStub('fs');
+    const onRequireFsPromisesStub = makeOnRequiresStub('fs-promises');
+    const onRequireCodecovStub = makeOnRequiresStub('codecov');
+    const onRequireCodecovLibStub = makeOnRequiresStub('codecov-lib');
+    const onRequireCpxStub = makeOnRequiresStub('cpx');
+    const onRequireCpxLibStub = makeOnRequiresStub('cpx-lib');
+
+    before(() => {
+      requireInTheMiddleSingleton.register('fs', onRequireFsStub);
+      requireInTheMiddleSingleton.register('fs/promises', onRequireFsPromisesStub);
+      requireInTheMiddleSingleton.register('codecov', onRequireCodecovStub);
+      requireInTheMiddleSingleton.register('codecov/lib/codecov.js', onRequireCodecovLibStub);
+      requireInTheMiddleSingleton.register('cpx', onRequireCpxStub);
+      requireInTheMiddleSingleton.register('cpx/lib/copy-sync.js', onRequireCpxLibStub);
+    });
+
+    beforeEach(() => {
+      onRequireFsStub.resetHistory();
+      onRequireFsPromisesStub.resetHistory();
+      onRequireCodecovStub.resetHistory();
+      onRequireCodecovLibStub.resetHistory();
+      onRequireCpxStub.resetHistory();
+      onRequireCpxLibStub.resetHistory();
+    });
+
+    it('should return a hooked object', () => {
+      const moduleName = 'm';
+      const onRequire = makeOnRequiresStub('m');
+      const hooked = requireInTheMiddleSingleton.register(moduleName, onRequire);
+      assert.deepStrictEqual(hooked, { moduleName, onRequire });
+    });
+
+    describe('core module', () => {
+      describe('AND module name matches', () => {
+        it('should call `onRequire`', () => {
+          const exports = require('fs');
+          assert.deepStrictEqual(exports.__ritmOnRequires, ['fs']);
+          sinon.assert.calledOnceWithExactly(onRequireFsStub, exports, 'fs', undefined);
+          sinon.assert.notCalled(onRequireFsPromisesStub);
+        });
+      });
+      describe('AND module name does not match', () => {
+        it('should not call `onRequire`', () => {
+          const exports = require('crypto');
+          assert.equal(exports.__ritmOnRequires, undefined);
+          sinon.assert.notCalled(onRequireFsStub);
+        });
+      });
+    });
+
+    describe('core module with sub-path', () => {
+      describe('AND module name matches', () => {
+        it('should call `onRequire`', () => {
+          const exports = require('fs/promises');
+          assert.deepStrictEqual(exports.__ritmOnRequires, ['fs', 'fs-promises']);
+          sinon.assert.calledOnceWithExactly(onRequireFsPromisesStub, exports, 'fs/promises', undefined);
+          sinon.assert.calledOnceWithMatch(onRequireFsStub, { __ritmOnRequires: ['fs', 'fs-promises'] }, 'fs/promises', undefined);
+        });
+      });
+    });
+
+    describe('non-core module', () => {
+      describe('AND module name matches', () => {
+        const baseDir = path.dirname(require.resolve('codecov'));
+        const modulePath = path.join('codecov', 'lib', 'codecov.js');
+        it('should call `onRequire`', () => {
+          const exports = require('codecov');
+          assert.deepStrictEqual(exports.__ritmOnRequires, ['codecov']);
+          sinon.assert.calledWithExactly(onRequireCodecovStub, exports, 'codecov', baseDir);
+          sinon.assert.calledWithMatch(onRequireCodecovStub, { __ritmOnRequires: ['codecov', 'codecov-lib'] }, modulePath, baseDir);
+          sinon.assert.calledWithMatch(onRequireCodecovLibStub, { __ritmOnRequires: ['codecov', 'codecov-lib'] }, modulePath, baseDir);
+        }).timeout(30000);
+      });
+    });
+
+    describe('non-core module with sub-path', () => {
+      describe('AND module name matches', () => {
+        const baseDir = path.resolve(path.dirname(require.resolve('cpx')), '..');
+        const modulePath = path.join('cpx', 'lib', 'copy-sync.js');
+        it('should call `onRequire`', () => {
+          const exports = require('cpx/lib/copy-sync');
+          assert.deepStrictEqual(exports.__ritmOnRequires, ['cpx', 'cpx-lib']);
+          sinon.assert.calledWithMatch(onRequireCpxStub, { __ritmOnRequires: ['cpx', 'cpx-lib'] }, modulePath, baseDir);
+          sinon.assert.calledWithExactly(onRequireCpxStub, exports, modulePath, baseDir);
+          sinon.assert.calledWithExactly(onRequireCpxLibStub, exports, modulePath, baseDir);
+        });
+      });
+    });
+  });
+});