We describe a highly sensitive flow cytometry-based CTL assay using the cleavage of caspase 3 in target cells as a readout. The assay involved labeling of cells with a cell tracker dye and staining permeabilized cells with an antibody recognizing cleaved caspase 3. The assay proved to be robust and reliable in measuring antigen-specific CTL activity in a number of human and murine systems, including MLR, human peptide-specific T-cell responses induced in vitro, and CTL responses following immunization of mice with viral and peptide vaccines. The assay was found to yield comparable results as 51Cr-release, but with markedly higher sensitivity. When compared to detection of antigen-specific T cells via HLA tetramer/pentamer-based methods of T-cell staining in HIV gag peptide-specific human T cell lines the caspase 3 cleavage readout assay exhibited a comparable level of sensitivity with detection of CTL function at antigen-specific T-cell frequencies of 1:15,000 or lower. A similar level of sensitivity was obtained when murine CTL assays were performed with MLR in which effector cells were highly diluted with naïve syngeneic spleen cells. Our results indicate that the caspase 3 cleavage assay may be a powerful tool to measure antigen-specific CTL responses in human vaccine trials and in pre-clinical animal models of CTL function at both high and low effector cell frequencies.