The comet assay (single cell gel electrophoresis) is the most common method for measuring DNA damage in eukaryotic cells or disaggregated tissues. The assay depends on the relaxation of supercoiled DNA in agarose-embedded nucleoids (the residual bodies remaining after lysis of cells with detergent and high salt), which allows the DNA to be drawn out towards the anode under electrophoresis, forming comet-like images as seen under fluorescence microscopy. The relative amount of DNA in the comet tail indicates DNA break frequency. The assay has been modified to detect various base alterations, by including digestion of nucleoids with a lesion-specific endonuclease. We describe here recent technical developments, theoretical aspects, limitations as well as advantages of the assay, and modifications to measure cellular antioxidant status and different types of DNA repair. We briefly describe the applications of this method in genotoxicity testing, human biomonitoring, and ecogenotoxicology.