Clostridial toxin substrates comprising a fluorescent donor, an acceptor, a Clostridial toxin recognition sequence including a cleavage site and a membrane targeting domain; methods for determining the activity of a Clostridial toxin from a test sample using such Clostridial toxin substrates; cell compositions comprising such Clostridial toxin substrates and a Clostridial toxin receptor; and methods for determining the activity of a Clostridial toxin from a test sample using such cell compositions.