diff --git a/.changes/validate-isolation-hook-set.md b/.changes/validate-isolation-hook-set.md
new file mode 100644
index 000000000..824783240
--- /dev/null
+++ b/.changes/validate-isolation-hook-set.md
@@ -0,0 +1,5 @@
+---
+"tauri-codegen": patch
+---
+
+Validate `__TAURI_ISOLATION_HOOK__` being set by a file in the isolation application.
diff --git a/core/tauri-codegen/src/context.rs b/core/tauri-codegen/src/context.rs
index 202b19cca..75aa8926b 100644
--- a/core/tauri-codegen/src/context.rs
+++ b/core/tauri-codegen/src/context.rs
@@ -330,13 +330,28 @@ pub fn context_codegen(data: ContextData) -> Result<TokenStream, EmbeddedAssetsE
       let dir = config_parent.join(dir);
       if !dir.exists() {
         panic!(
-          "The isolation dir configuration is set to `{:?}` but this path doesn't exist",
+          "The isolation application path is set to `{:?}` but it does not exist",
           dir
         )
       }
 
+      let mut sets_isolation_hook = false;
+
       let key = uuid::Uuid::new_v4().to_string();
-      let assets = EmbeddedAssets::new(dir.clone(), &options, map_isolation(&options, dir))?;
+      let map_isolation = map_isolation(&options, dir.clone());
+      let assets = EmbeddedAssets::new(dir, &options, |key, path, input, csp_hashes| {
+        // we check if `__TAURI_ISOLATION_HOOK__` exists in the isolation code
+        // before modifying the files since we inject our own `__TAURI_ISOLATION_HOOK__` reference in HTML files
+        if String::from_utf8_lossy(input).contains("__TAURI_ISOLATION_HOOK__") {
+          sets_isolation_hook = true;
+        }
+        map_isolation(key, path, input, csp_hashes)
+      })?;
+
+      if !sets_isolation_hook {
+        panic!("The isolation application does not contain a file setting the `window.__TAURI_ISOLATION_HOOK__` value.");
+      }
+
       let schema = options.isolation_schema;
 
       quote!(#root::Pattern::Isolation {
diff --git a/core/tauri-codegen/src/embedded_assets.rs b/core/tauri-codegen/src/embedded_assets.rs
index d930b7700..a5983fa6e 100644
--- a/core/tauri-codegen/src/embedded_assets.rs
+++ b/core/tauri-codegen/src/embedded_assets.rs
@@ -246,7 +246,12 @@ impl EmbeddedAssets {
   pub fn new(
     input: impl Into<EmbeddedAssetsInput>,
     options: &AssetOptions,
-    map: impl Fn(&AssetKey, &Path, &mut Vec<u8>, &mut CspHashes) -> Result<(), EmbeddedAssetsError>,
+    mut map: impl FnMut(
+      &AssetKey,
+      &Path,
+      &mut Vec<u8>,
+      &mut CspHashes,
+    ) -> Result<(), EmbeddedAssetsError>,
   ) -> Result<Self, EmbeddedAssetsError> {
     // we need to pre-compute all files now, so that we can inject data from all files into a few
     let RawEmbeddedAssets { paths, csp_hashes } = RawEmbeddedAssets::new(input.into(), options)?;
@@ -262,7 +267,8 @@ impl EmbeddedAssets {
         assets: HashMap::new(),
       },
       move |mut state, (prefix, entry)| {
-        let (key, asset) = Self::compress_file(&prefix, entry.path(), &map, &mut state.csp_hashes)?;
+        let (key, asset) =
+          Self::compress_file(&prefix, entry.path(), &mut map, &mut state.csp_hashes)?;
         state.assets.insert(key, asset);
         Result::<_, EmbeddedAssetsError>::Ok(state)
       },
@@ -292,7 +298,12 @@ impl EmbeddedAssets {
   fn compress_file(
     prefix: &Path,
     path: &Path,
-    map: &impl Fn(&AssetKey, &Path, &mut Vec<u8>, &mut CspHashes) -> Result<(), EmbeddedAssetsError>,
+    map: &mut impl FnMut(
+      &AssetKey,
+      &Path,
+      &mut Vec<u8>,
+      &mut CspHashes,
+    ) -> Result<(), EmbeddedAssetsError>,
     csp_hashes: &mut CspHashes,
   ) -> Result<Asset, EmbeddedAssetsError> {
     let mut input = std::fs::read(path).map_err(|error| EmbeddedAssetsError::AssetRead {