Mca-PLGL-Dap(Dnp)-AR-NH2 is a sensitive synthetic peptide FRET substrate for the determination of matrix metalloproteinase (MMP) activity. Protease cleavage at the -Gly-Leu- bond releases the highly fluorescent 7-methoxycoumarin (Mca) group from the influence of the 2,4-dinitrophenyl (Dnp) internal quencher, resulting in a 190 fold increase in fluorescence intensity. Mca-PLGL-Dap(Dnp)-AR-NH2 is suitable for use in continuous assays at enzyme concentrations comparable to those used with macromolecular substrates and also for the assessment of total matrix metalloproteinase in crude preparations.