Ex vivo detection of virus-specific cytotoxic T lymphocyte (CTL) responses is limited to the use of methods assessing cytokine production, degranulation, or perforin contents of antigen-specific CD8+ T cells. Generally, their cytotoxic activity is detectable only after cultivation. We describe the fluorescent antigen–transfected target cell–CTL (FATT-CTL) assay, which measures antigen-specific cytotoxicity ex vivo. Target cells were generated by nucleofection with DNA vectors encoding antigen–green fluorescent protein (GFP) fusion proteins. After coculture at various effector:target (E:T) cell ratios, viable and dead GFP-positive cells were quantified by flow cytometry, and antigen-specific target-cell elimination was calculated. The assay was validated with human immunodeficiency virus (HIV)– and influenza virus–specific CTL clones and revealed cytotoxicity at lower E:T cell ratios than standard 51Cr-release assays. Moreover, antigen-specific cytotoxicity was detected ex vivo within 1 day in peripheral blood mononuclear cells from HIV-infected individuals. The FATT-CTL assay provides a versatile tool that will advance our understanding of cell-mediated immunity