We report here the development of the polymerase inhibition assay (PI assay), a methodology capable of simultaneously identifying multiple DNA-damaging agents. The PI assay was developed in order to fulfil a requirement for the screening of new pharmaceuticals for potential DNA-damaging effects. The assay has the potential to screen hundreds of new compounds per week because of the microtiter plate format employed. We review previous descriptions of the phenomenon and provide researchers with the necessary methodology to obtain optimum polymerase inhibition effects. The assay is based on the inhibition of DNA polymerases (including those used in the polymerase chain reaction (PCR)) encountering damaged DNA bases. Hence, DNA-damaging agents can be identified by a corresponding reduction in PCR amplification after exposure. We demonstrate the detection of polymerase inhibition induced by a range of model genotoxic agents (N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea, and ultraviolet (UV) C radiation), illustrating the successful application of the methodology. In addition, the PI assay is shown to be capable of detecting DNA damaging agents of biological relevance, i.e., known human carcinogens. These were N-OH-PhIP (from cooked meat) and UV-B (from sunlight). In addition to its employment in the detection of putative DNA damaging agents, the PI assay may also be applied as a research tool in carcinogenicity studies.