Lymphocyte-mediated cytotoxicity via granule exocytosis operates by the perforin-mediated transfer of granzymes from CTLs and NK cells into target cells where caspase activation and other death pathways are triggered. Granzyme B (GzB) is a major cytotoxic effector in this pathway, and its fate in target cells has been studied by several groups using immunodetection. In this study, we have used a newly developed cell-permeable fluorogenic GzB substrate to measure this protease activity in three different living targets following contact with cytotoxic effectors. Although no GzB activity is measurable in CTL or NK92 effector cells, this activity rapidly becomes detectable throughout the target cytoplasm after effector-target engagement. We have combined the GzB substrate with a second fluorogenic substrate selective for caspase 3 to allow both flow cytometry and fluorescence confocal microscopy studies of cytotoxicity. With both effectors, caspase 3 activity appears subsequent to that of GzB inside all three targets. Overexpression of Bcl-2 in target cells has minimal effects on lysis, NK- or CTL-delivered GzB activity, or activation of target caspase 3. Detection of target GzB activity followed by caspase 3 activation provides a unique readout of a potentially lethal injury delivered by cytotoxic lymphocytes.