diff --git a/instrumentation/opentelemetry-instrumentation-fastapi/src/opentelemetry/instrumentation/fastapi/__init__.py b/instrumentation/opentelemetry-instrumentation-fastapi/src/opentelemetry/instrumentation/fastapi/__init__.py
index 97854830c2..23f6470ef7 100644
--- a/instrumentation/opentelemetry-instrumentation-fastapi/src/opentelemetry/instrumentation/fastapi/__init__.py
+++ b/instrumentation/opentelemetry-instrumentation-fastapi/src/opentelemetry/instrumentation/fastapi/__init__.py
@@ -191,7 +191,7 @@ def client_response_hook(span: Span, scope: dict[str, Any], message: dict[str, A
 import fastapi
 from starlette.applications import Starlette
 from starlette.middleware.errors import ServerErrorMiddleware
-from starlette.routing import Match
+from starlette.routing import Match, Route
 from starlette.types import ASGIApp, Receive, Scope, Send
 
 from opentelemetry.instrumentation._semconv import (
@@ -474,7 +474,11 @@ def _get_route_details(scope):
     route = None
 
     for starlette_route in app.routes:
-        match, _ = starlette_route.matches(scope)
+        match, _ = (
+            Route.matches(starlette_route, scope)
+            if isinstance(starlette_route, Route)
+            else starlette_route.matches(scope)
+        )
         if match == Match.FULL:
             try:
                 route = starlette_route.path
diff --git a/instrumentation/opentelemetry-instrumentation-fastapi/tests/test_fastapi_instrumentation.py b/instrumentation/opentelemetry-instrumentation-fastapi/tests/test_fastapi_instrumentation.py
index a97697f582..661c7097cd 100644
--- a/instrumentation/opentelemetry-instrumentation-fastapi/tests/test_fastapi_instrumentation.py
+++ b/instrumentation/opentelemetry-instrumentation-fastapi/tests/test_fastapi_instrumentation.py
@@ -20,14 +20,17 @@
 import weakref as _weakref
 from contextlib import ExitStack
 from timeit import default_timer
-from typing import Any, cast
+from typing import Any, Final, cast
 from unittest.mock import Mock, call, patch
 
 import fastapi
 import pytest
 from fastapi.middleware.httpsredirect import HTTPSRedirectMiddleware
 from fastapi.responses import JSONResponse, PlainTextResponse
+from fastapi.routing import APIRoute
 from fastapi.testclient import TestClient
+from starlette.routing import Match
+from starlette.types import Receive, Scope, Send
 
 import opentelemetry.instrumentation.fastapi as otel_fastapi
 from opentelemetry import trace
@@ -131,6 +134,23 @@
 )
 
 
+class CustomMiddleware:
+    def __init__(self, app: fastapi.FastAPI) -> None:
+        self.app = app
+
+    async def __call__(
+        self, scope: Scope, receive: Receive, send: Send
+    ) -> None:
+        scope["nonstandard_field"] = "here"
+        await self.app(scope, receive, send)
+
+
+class CustomRoute(APIRoute):
+    def matches(self, scope: Scope) -> tuple[Match, Scope]:
+        assert "nonstandard_field" in scope
+        return super().matches(scope)
+
+
 class TestBaseFastAPI(TestBase):
     def _create_app(self):
         app = self._create_fastapi_app()
@@ -191,6 +211,7 @@ def setUp(self):
         self._instrumentor = otel_fastapi.FastAPIInstrumentor()
         self._app = self._create_app()
         self._app.add_middleware(HTTPSRedirectMiddleware)
+        self._app.add_middleware(CustomMiddleware)
         self._client = TestClient(self._app, base_url="https://testserver:443")
         # run the lifespan, initialize the middleware stack
         # this is more in-line with what happens in a real application when the server starts up
@@ -210,6 +231,7 @@ def tearDown(self):
     def _create_fastapi_app():
         app = fastapi.FastAPI()
         sub_app = fastapi.FastAPI()
+        custom_router = fastapi.APIRouter(route_class=CustomRoute)
 
         @sub_app.get("/home")
         async def _():
@@ -235,6 +257,12 @@ async def _():
         async def _():
             raise UnhandledException("This is an unhandled exception")
 
+        @custom_router.get("/success")
+        async def _():
+            return None
+
+        app.include_router(custom_router, prefix="/custom-router")
+
         app.mount("/sub", app=sub_app)
         app.host("testserver2", sub_app)
 
@@ -313,6 +341,28 @@ def test_sub_app_fastapi_call(self):
             span.attributes[HTTP_URL],
         )
 
+    def test_custom_api_router(self):
+        """
+        This test is to ensure that custom API routers the OpenTelemetryMiddleware does not cause issues with
+        custom API routers that depend on non-standard fields on the ASGI scope.
+        """
+        resp: Final = self._client.get("/custom-router/success")
+        spans: Final = self.memory_exporter.get_finished_spans()
+        spans_with_http_attributes = [
+            span
+            for span in spans
+            if (HTTP_URL in span.attributes or HTTP_TARGET in span.attributes)
+        ]
+        self.assertEqual(200, resp.status_code)
+        for span in spans_with_http_attributes:
+            self.assertEqual(
+                "/custom-router/success", span.attributes[HTTP_TARGET]
+            )
+            self.assertEqual(
+                "https://testserver/custom-router/success",
+                span.attributes[HTTP_URL],
+            )
+
     def test_host_fastapi_call(self):
         client = TestClient(self._app, base_url="https://testserver2:443")
         client.get("/")
@@ -1017,6 +1067,7 @@ def test_metric_uninstrument(self):
     def _create_fastapi_app():
         app = fastapi.FastAPI()
         sub_app = fastapi.FastAPI()
+        custom_router = fastapi.APIRouter(route_class=CustomRoute)
 
         @sub_app.get("/home")
         async def _():
@@ -1042,6 +1093,12 @@ async def _():
         async def _():
             raise UnhandledException("This is an unhandled exception")
 
+        @custom_router.get("/success")
+        async def _():
+            return None
+
+        app.include_router(custom_router, prefix="/custom-router")
+
         app.mount("/sub", app=sub_app)
 
         return app