Duplicate the database - label, name or place the duplicate to identify it, Open it and delete the layout. Test everything.
If you have FileMaker advanced, you can generate a database design report and do a text search on it for you layout name to see each place in your system that refers to it so that you can find and update it to refer to a layout for the same table (may need to check on table occurrences, not just tables here.) so that you can safely delete it.
There are also third party development tools out there that can likely assist you in such operations.