Cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells carry out an important part of the immune response by eliminating virus-infected and malignant cells. Cytotoxicity is usually quantified by measuring the release of radioactive chromium (Cr⁵¹) or cytosolic enzymes like lactate dehydrogenase (LDH) from the dying target cells. One of the common drawbacks of these assays is that cytotoxicity can be measured only at the end-point thus information regarding the kinetics of killing is lost. We developed an assay for cytotoxicity which allows us to monitor and quantify the killing kinetics in real-time. Target cells were pre-labelled with the fluorescent dye calcein-AM and killer cells were added at the beginning of the experiment. Fluorescence was collected from either single target cells with a microscope or from a whole target cell population using a fluorescence microplate reader. After receiving the lethal hit, dying target cells rapidly lose their fluorescence, which allows us to follow the killing process with a high time resolution. The number of surviving target cells follows an exponential function with characteristic time constants depending on (1) the type of killer cell, (2) the cell density and (3) the ratio of killer to target cell numbers. The assay was tested successfully with activated primary human CD8⁺ cells, primary human NK cells and the human cell line TALL-104, and killing time constants were 48, 19.5 and 17 minutes (E:T 10:1), respectively. We believe that this technique allows a better insight into the mechanisms of cytotoxicity.