diff --git a/ui/web.go b/ui/web.go
index 3b81135237..4c6d189463 100644
--- a/ui/web.go
+++ b/ui/web.go
@@ -1,4 +1,4 @@
-// Copyright 2015 Prometheus Team
+// Copyright The Prometheus 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
@@ -19,6 +19,7 @@ import (
 	"net/http"
 	_ "net/http/pprof" // Comment this line to disable pprof endpoint.
 	"path"
+	"strings"
 
 	"github.com/prometheus/client_golang/prometheus/promhttp"
 	"github.com/prometheus/common/route"
@@ -87,8 +88,17 @@ func Register(r *route.Router, reloadCh chan<- chan error, logger *slog.Logger)
 		w.WriteHeader(http.StatusOK)
 	})
 
-	r.Get("/debug/*subpath", http.DefaultServeMux.ServeHTTP)
-	r.Post("/debug/*subpath", http.DefaultServeMux.ServeHTTP)
+	debugHandlerFunc := func(w http.ResponseWriter, req *http.Request) {
+		subpath := route.Param(req.Context(), "subpath")
+		req.URL.Path = path.Join("/debug", subpath)
+		// path.Join removes trailing slashes, but some pprof handlers expect them.
+		if strings.HasSuffix(subpath, "/") && !strings.HasSuffix(req.URL.Path, "/") {
+			req.URL.Path += "/"
+		}
+		http.DefaultServeMux.ServeHTTP(w, req)
+	}
+	r.Get("/debug/*subpath", debugHandlerFunc)
+	r.Post("/debug/*subpath", debugHandlerFunc)
 }
 
 func disableCaching(w http.ResponseWriter) {
diff --git a/ui/web_test.go b/ui/web_test.go
new file mode 100644
index 0000000000..e6293d1834
--- /dev/null
+++ b/ui/web_test.go
@@ -0,0 +1,61 @@
+// Copyright The Prometheus 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
+//
+// http://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.
+
+package ui
+
+import (
+	"log/slog"
+	"net/http"
+	"net/http/httptest"
+	"os"
+	"testing"
+
+	"github.com/prometheus/common/route"
+	"github.com/stretchr/testify/require"
+)
+
+func TestDebugHandlersWithRoutePrefix(t *testing.T) {
+	logger := slog.New(slog.NewTextHandler(os.Stderr, nil))
+	reloadCh := make(chan chan error)
+
+	// Test with route prefix
+	routePrefix := "/prometheus/alertmanager"
+	router := route.New().WithPrefix(routePrefix)
+	Register(router, reloadCh, logger)
+
+	// Test GET request to pprof index (note: pprof index returns text/html)
+	req := httptest.NewRequest("GET", routePrefix+"/debug/pprof/", nil)
+	w := httptest.NewRecorder()
+	router.ServeHTTP(w, req)
+
+	require.Equal(t, http.StatusOK, w.Code)
+	require.Contains(t, w.Body.String(), "/debug/pprof/", "pprof page did not load with expected content when using a route prefix")
+
+	// Test GET request to pprof heap endpoint
+	req = httptest.NewRequest("GET", routePrefix+"/debug/pprof/heap", nil)
+	w = httptest.NewRecorder()
+	router.ServeHTTP(w, req)
+
+	require.Equal(t, http.StatusOK, w.Code)
+
+	// Test without route prefix (should also work)
+	router2 := route.New()
+	Register(router2, reloadCh, logger)
+
+	req = httptest.NewRequest("GET", "/debug/pprof/", nil)
+	w = httptest.NewRecorder()
+	router2.ServeHTTP(w, req)
+
+	require.Equal(t, http.StatusOK, w.Code)
+	require.Contains(t, w.Body.String(), "/debug/pprof/", "pprof page did not load with expected content")
+}