Fluorogenic synthetic substrates are commonly used to monitor the activity of peptidases in vitro. This unit presents a representative protocol that employs (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L-2,3-diaminopropionyl)-Ala-Arg-NH2 (Mca-Pro-Leu-Gly~Leu-Dpa-Ala-Arg-NH2) as a substrate to assay matrix metallopeptidases (MMPs). This substrate was first described for the assay of MMP-1, -2 and -3 and it is now widely used as a general MMP substrate. Protocols are given for both stopped-time assays (suitable for assaying MMP activity in a large number of samples) and continuous assays (commonly used when establishing an assay protocol or investigating kinetic aspects of enzyme behavior). Other fluorogenic peptides and protein substrates, together with non-fluorogenic alternatives, are also discussed.