diff --git a/packages/angular_devkit/build_angular/src/builders/karma/application_builder.ts b/packages/angular_devkit/build_angular/src/builders/karma/application_builder.ts
index 5c59ae05375b..f6b2e8b944cd 100644
--- a/packages/angular_devkit/build_angular/src/builders/karma/application_builder.ts
+++ b/packages/angular_devkit/build_angular/src/builders/karma/application_builder.ts
@@ -186,12 +186,7 @@ async function collectEntrypoints(
     projectSourceRoot,
   );
 
-  const entryPoints = new Set([
-    ...testFiles,
-    '@angular-devkit/build-angular/src/builders/karma/init_test_bed.js',
-  ]);
-
-  return entryPoints;
+  return new Set(testFiles);
 }
 
 async function initializeApplication(
@@ -218,6 +213,14 @@ async function initializeApplication(
     fs.rm(outputPath, { recursive: true, force: true }),
   ]);
 
+  let mainName = 'init_test_bed';
+  if (options.main) {
+    entryPoints.add(options.main);
+    mainName = path.basename(options.main, path.extname(options.main));
+  } else {
+    entryPoints.add('@angular-devkit/build-angular/src/builders/karma/init_test_bed.js');
+  }
+
   const instrumentForCoverage = options.codeCoverage
     ? createInstrumentationFilter(
         projectSourceRoot,
@@ -261,10 +264,19 @@ async function initializeApplication(
   karmaOptions.files.push(
     // Serve polyfills first.
     { pattern: `${outputPath}/polyfills.js`, type: 'module' },
-    // Allow loading of chunk-* files but don't include them all on load.
-    { pattern: `${outputPath}/{chunk,worker}-*.js`, type: 'module', included: false },
+    // Serve global setup script.
+    { pattern: `${outputPath}/${mainName}.js`, type: 'module' },
+    // Serve all source maps.
+    { pattern: `${outputPath}/*.map`, included: false },
   );
 
+  if (hasChunkOrWorkerFiles(buildOutput.files)) {
+    karmaOptions.files.push(
+      // Allow loading of chunk-* files but don't include them all on load.
+      { pattern: `${outputPath}/{chunk,worker}-*.js`, type: 'module', included: false },
+    );
+  }
+
   karmaOptions.files.push(
     // Serve remaining JS on page load, these are the test entrypoints.
     { pattern: `${outputPath}/*.js`, type: 'module' },
@@ -316,6 +328,12 @@ async function initializeApplication(
   return [karma, parsedKarmaConfig, buildOptions];
 }
 
+function hasChunkOrWorkerFiles(files: Record<string, unknown>): boolean {
+  return Object.keys(files).some((filename) => {
+    return /(?:^|\/)(?:worker|chunk)[^/]+\.js$/.test(filename);
+  });
+}
+
 export async function writeTestFiles(files: Record<string, ResultFile>, testDir: string) {
   const directoryExists = new Set<string>();
   // Writes the test related output files to disk and ensures the containing directories are present
diff --git a/packages/angular_devkit/build_angular/src/builders/karma/tests/behavior/fake-async_spec.ts b/packages/angular_devkit/build_angular/src/builders/karma/tests/behavior/fake-async_spec.ts
index a6cde25eb435..463dd625ac7f 100644
--- a/packages/angular_devkit/build_angular/src/builders/karma/tests/behavior/fake-async_spec.ts
+++ b/packages/angular_devkit/build_angular/src/builders/karma/tests/behavior/fake-async_spec.ts
@@ -9,7 +9,7 @@
 import { execute } from '../../index';
 import { BASE_OPTIONS, KARMA_BUILDER_INFO, describeKarmaBuilder } from '../setup';
 
-describeKarmaBuilder(execute, KARMA_BUILDER_INFO, (harness, setupTarget, isApp) => {
+describeKarmaBuilder(execute, KARMA_BUILDER_INFO, (harness, setupTarget) => {
   describe('Behavior: "fakeAsync"', () => {
     beforeEach(async () => {
       await setupTarget(harness);
diff --git a/packages/angular_devkit/build_angular/src/builders/karma/tests/options/main_spec.ts b/packages/angular_devkit/build_angular/src/builders/karma/tests/options/main_spec.ts
new file mode 100644
index 000000000000..104b15cec31c
--- /dev/null
+++ b/packages/angular_devkit/build_angular/src/builders/karma/tests/options/main_spec.ts
@@ -0,0 +1,51 @@
+/**
+ * @license
+ * Copyright Google LLC All Rights Reserved.
+ *
+ * Use of this source code is governed by an MIT-style license that can be
+ * found in the LICENSE file at https://angular.dev/license
+ */
+
+import { execute } from '../../index';
+import { BASE_OPTIONS, KARMA_BUILDER_INFO, describeKarmaBuilder } from '../setup';
+
+describeKarmaBuilder(execute, KARMA_BUILDER_INFO, (harness, setupTarget) => {
+  describe('Option: "main"', () => {
+    beforeEach(async () => {
+      await setupTarget(harness);
+    });
+
+    beforeEach(async () => {
+      await harness.writeFiles({
+        'src/magic.ts': `Object.assign(globalThis, {MAGIC_IS_REAL: true});`,
+        'src/magic.spec.ts': `
+            declare const MAGIC_IS_REAL: boolean;
+            describe('Magic', () => {
+              it('can be scientificially proven to be true', () => {
+                expect(typeof MAGIC_IS_REAL).toBe('boolean');
+              });
+            });`,
+      });
+      // Remove this test, we don't expect it to pass with our setup script.
+      await harness.removeFile('src/app/app.component.spec.ts');
+
+      // Add src/magic.ts to tsconfig.
+      interface TsConfig {
+        files: string[];
+      }
+      const tsConfig = JSON.parse(harness.readFile('src/tsconfig.spec.json')) as TsConfig;
+      tsConfig.files.push('magic.ts');
+      await harness.writeFile('src/tsconfig.spec.json', JSON.stringify(tsConfig));
+    });
+
+    it('uses custom setup file', async () => {
+      harness.useTarget('test', {
+        ...BASE_OPTIONS,
+        main: './src/magic.ts',
+      });
+
+      const { result } = await harness.executeOnce();
+      expect(result?.success).toBeTrue();
+    });
+  });
+});