Activation of ICE-family proteases/caspases initiates apoptosis in mammalian cells. The Caspase-9 Fluorometric Assay Kit provides a simple and convenient means for assaying the activity of caspases that recognize the sequence LEHD. The assay is based on detection of cleavage of substrate LEHD-AFC (AFC: 7-amino-4-trifluoromethyl coumarin). LEHD-AFC emits blue light (λmax = 400 nm); upon cleavage of the substrate by caspase-9 or related caspases, free AFC emits a yellow-green fluorescence (λmax = 505 nm), which can be quantified using a fluorometer or fluorecence microtiter plate reader.