The light chain of tetanus toxin (TeNT-L chain), endowed with a zinc metalloendopeptidase activity, cleaves specifically the vesicle-associated membrane protein (VAMP), also called synaptobrevin, at a single peptide bond (Gln76-Phe77), resulting in the blockade of neuroexocytosis. The 50-mer synaptobrevin peptide S 39-88, synthesized by solid-phase peptide synthesis, was determined to be the minimum substrate of TeNT still notably hydrolyzed by TeNT-L chain. In this peptide, Tyr88 was substituted by the highly fluorescent amino acid (L) pyrenylalanine (Pya) which was synthesized in good yields by an enantioselective method. The fluorescent substrate [Pya88] S 39-88 was cleaved four times more rapidly by TeNT-L chain than S 39-88 (kcat/Km = 9635 and 2455 M-1.min-1, respectively). One of the two metabolites formed by the action of TeNT L chain, [Pya88] S 77-88, was easily separated from the substrate in one step using Sep-Pak Vac C18 cartridges and its concentration quantified by fluorescence. This novel enzymatic assay, which could be easily extended to other clostridial neurotoxins, is a major improvement in term of sensitivity and time saving, compared to currently used methods (SDS-PAGE, HPLC). It lends itself readily to automation for large-scale screening of selective and potent inhibitors of these neurotoxins which remain to be developed.