Abstract: The comet assay (single-cell gel electrophoresis) is a simple method for measuring DNA strand breaks in cells that are embedded in agarose and lysed to remove membranes and soluble cell constituents (including most histones). The assay depends on the ability of breaks to relax supercoiling, which allows DNA still attached to a nuclear matrix to move toward the anode under electrophoresis, forming a `comet tail¿ when viewed by fluorescence microscopy. The percentage of DNA in the tail indicates the frequency of DNA breaks. An additional step ¿ digestion with lesion-specific endonucleases ¿ is introduced after lysis in order to detect different kinds of DNA damage. The assay can be calibrated to give quantitative measures of DNA damage. It has been widely used in genotoxicity testing and human biomonitoring, and also in ecogenotoxicology, as well as in basic research. We here discuss the development of the method, its principles, applications, and limitations, and attempt to dispel some fallacies that trouble the assay. We provide a detailed protocol, including a recent modification that increases the assay's throughput.