Abstract DNA lesions in mammalian cells may be induced by reactive oxygen species, alkylation, and ionizing radiation. This damage can then be repaired via the base excision repair (BER) pathway, which includes single strand break repair (SSBR). Thus, the BER (SSBR) pathway plays a critical role in maintaining genomic integrity, and may help us to better understand the mechanisms of aging, tumor formation, and degenerative diseases. AP site (apurinic/apyrimidinic site) or damaged base excision, nucleotide insertion and ligation are included in the BER (SSBR) pathway, which are facilitated by different enzymes at each step. Most previous in vitro BER studies have used modified radiolabeled 32P oligonucleotide substrates. Which is a very conventional method for in vitro BER assay. However, the use of radioactive isotope material was limited in various laboratories which are unable to handle radioactive hazard. In this study, we developed a novel technique using real-time quantitative PCR (qPCR) to quantify BER activity in in vitro assays. Single strand breaks, DNA ligase activity, and glycosylase activity were detected to establish the feasibility and advantages of this qPCR technique for in vitro BER profiling.