<p>The thiopurines 6-mercaptopurine and 6-thioguanine (TG) are analogs of guanine and are used in the treatment of hematological malignancies and immune-mediated inflammatory diseases. The mechanism of action includes the incorporation of TG into DNA (DNA-TG) in competition with natural guanine. DNA-TG can undergo S⁶-methylation (DNA-MeTG), and O⁶-methylguanine methyltransferase can remove alkyl groups from O⁶-alkylguanine, but is less effective with DNA-MeTG, which favors S⁶-MeTG·T mismatching. If unrecognized by the post-replicative mismatch repair system (MMR), this can lead to an increased mutational load with the formation of neoepitopes, which can potentially increase tumor cell recognition by the immune system. The exact role of DNA-MeTG in the antileukemic efficacy of thiopurines remains to be elucidated. In this work, an LC-MS/MS method for the quantitation of 6-thio-2′-deoxyguanosine (dTG) and its methylated metabolite 6-methylthio-2′-deoxyguanosine (dMeTG) in genomic DNA using enzymatically synthesized isotope-labeled internal standards 6-thio-2′-deoxyguanosine-¹³C₂¹⁵N and 6-methylthio-2′-deoxyguanosine-D₃ is presented. Purified DNA was enzymatically digested into nucleosides, from which dMeTG and dTG could be quantified with LC-MS/MS and normalized to the amount of 2′-deoxyguanosine in the DNA. The applicability of the method was demonstrated in Jurkat cells treated with TG, where dMeTG and dTG could be quantified in samples containing less than 2 µg DNA. dMeTG could also be detected in patient samples, although in low amounts and primarily in samples with high DNA-TG levels. The developed method for the quantitation of dMeTG and dTG can be used in further studies to investigate the role of DNA-MeTG in the mechanism of action of thiopurines, including its antileukemic efficacy and effects on acquired mutations.</p>