<span class="paragraphSection"><div class="boxTitle">Abstract</div>The nucleotide excision repair (NER) system removes UV photoproducts from the genome in the form of small, excised, damage-containing DNA oligonucleotides (sedDNAs). However, the methods that are traditionally used to detect these repair products in human cells have several limitations, including the presence of other contaminating large DNAs and labeling efficiency. We therefore developed a novel microplate-based competitive immunoassay platform to precisely measure sedDNAs containing pyrimidine (6–4) pyrimidone photoproducts [(6–4)PPs] and cyclobutane pyrimidine dimer (CPD) following UV exposure. The immunoassay employs polyethylene glycol to separate sedDNAs from larger contaminating DNA molecules along with immobilized UV-irradiated oligonucleotides and damage-specific antibodies. Our results demonstrate that this method is highly sensitive and specific, enabling the detection of NER-dependent sedDNAs generated by minimal UV doses and within a short time frame after UV exposure. Moreover, quantification of (6–4)PP- and CPD-containing sedDNAs in this assay allowed the detection of up to 22-fold more (6–4)PP-sedDNAs and up to 7-fold more CPD-sedDNAs than by conventional 3′-end labeling. Thus, this novel immunoassay shows remarkable sensitivity and accuracy for quantifying sedDNAs in UV-irradiated cells and provides a convenient platform for advancing our understanding of the molecular mechanisms of the NER system.</span>