Calpain, a cytosolic calcium-dependent cysteine protease, is involved in many forms of apoptosis. The Calpain Activity Assay Kit provides a simple and convenient means for assaying calpain activity. The assay is based on fluorometric detection of cleavage of calpain substrate Ac-LLY-AFC. Ac-LLY-AFC emits blue light (max = 400 nm); upon cleavage of the substrate by calpain or related proteases, free AFC emits a yellow-green fluorescence (max = 505 nm), which can be quantified using a fluorometer or a fluorescence plate reader. Comparison of the fluorescence of AFC from a treated sample with a normal control allows determination of the changes in calpain activity.