The calpains are a family of intracellular Ca2+ dependent cysteine proteases. The best-characterized calpains are isoforms 1 (μ) and 2 (m). They respond to Ca2+ signals by cleaving many specific proteins, thereby irreversibly modifying their function(s). Calpains 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. The SensoLyte® 520 Calpain Assay Kit is optimized for detecting calpain activity. This kit contains a novel internally quenched 5-FAM/QXL® 520 FRET substrate. Calpain protease cleaves the FRET substrate into two separate fragments resulting in the release of 5-FAM fluorescence which can be monitored at excitation/emission= 490/520 nm. Increase in fluorescence is proportional to the calpain activity. The assay can detect both calpain 1 (μ) and 2 (m) activities and is ideal for kinetic study of these enzymes. The long wavelength fluorescence of 5-FAM is less interfered by the autofluorescence of components in biological samples and test compounds. The assays are performed in a convenient 96-well microplate format.