Calpains are a family of intracellular Ca2+ dependent cysteine proteases. They respond to Ca2+ signals by cleaving many specific proteins, thereby irreversibly modifying their function(s). They are implicated in a variety of Ca2+ regulated cellular processes as well as various pathological phenomena, such as ischemic injury, muscular dystrophy, diabetes, cataract, atherosclerosis, Alzheimer’s disease, and cancer. Calpains represent potential therapeutic targets for drug discovery. This 7-amino-4-methylcoumarin (AMC) labeled peptide is widely used as a fluorogenic substrate for 20S proteasome, calpains and other chymotrypsin-like proteases. Cleavage of this AMC peptide by the enzymes generates strongly fluorescent AMC that is monitored fluorimetrically at Abs/Em=353/442 nm.