Single strand breaks (SSBs) are among the most frequent DNA lesions caused by endogenous and exogenous agents. The most utilized alkaline-based assays for SSB detection frequently give false positive results due to artifactual SSBs arising from cleaved alkali-labile sites. Here we developed a specific SSB assay using alkaline gel electrophoresis (AGE) coupled with an O-hydroxylamine, O-(Tetrahydro-2H-pyran-2-yl)hydroxylamine (OTX). OTX stabilizes AP sites to prevent their incision during alkaline DNA denaturation. DNA from DT40 and isogenic polymerase [beta] null cells exposed to methyl methanesulfonate were applied to the OTX-coupled AGE assay. The detection of true SSB formation was observed in each cell line with significantly greater formation observed in the null cells. Furthermore, a modification of the assay demonstrated the accumulation of intact AP sites in genomic DNA from both cell lines. OTX use represents a facile approach for assessing SSBs, whose benefits may also be applied to other established SSB assays.