A damaged DNA detection assay (3D assay) using plasmid DNA adsorbed on sensitized microplates as the substrate for an in vitro repair reaction is presented. DNA lesions are repaired by the excision repair pathway which implies an incision-excision reaction followed by DNA repair synthesis. In the 3D assay, we took advantage of (i) plasmid DNA adsorption on polylysine-coated microplates that allowed various DNA-damaging treatments; (ii) a protein extract that reproduced the repair reaction in vitro; (iii) incorporation of digoxigenylated deoxynucleotide monophosphate during the DNA polymerization step which was quantified by a chemiluminescent reaction. Under experimental conditions for quantitative DNA adsorption, a dose-response relationship between the extent of DNA modification and the repair synthesis activity was found. Optimization of the biochemical parameters with UVC light-induced DNA lesions allowed the detection of about one photoproduct per plasmid circle. This new assay that permits a quick and easy assessment of DNA damage is applicable to the screening of genotoxic compounds and to the testing of DNA-damaging treatments.