Quantification of DNA lesions constitutes one of the main tasks in toxicology and in assessing health risks accompanied by exposure to carcinogens. Tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) can undergo metabolic transformation to give a reactive intermediate that pyridyloxobutylates nucleobases and phosphate backbone of DNA. Here, we reported a highly sensitive method, relying on the use of nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry (nLC-nESI-MS/MS), for the simultaneous quantifications of O6-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O6-POBdG) as well as O2- and O4-[4-(3-pyridyl)-4-oxobut-1-yl]-thymidine (O2-POBdT and O4-POBdT). By using this method, we measured the levels of the three DNA adducts with the use of 10 μg of DNA isolated from cultured mammalian cells exposed to a model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc). Our results demonstrated, for the first time, the formation of O4-POBdT in naked DNA and in genomic DNA of cultured mammalian cells exposed with NNKOAc. We also revealed that the levels of the three lesions increased with the dose of NNKOAc and that O2-POBdT and O4-POBdT could be subjected to repair by the nucleotide excision repair (NER) pathway. The method reported here will be useful for investigations about the involvement of other DNA repair pathways in the removal of these lesions and for human toxicological studies in the future.