Exposure to reactive oxygen species can result in formation of oxidized nucleotides which increase the frequency of mutations, DNA damage, and cell death. MutT Homolog 1 (MTH1) is an enzyme that catalyzes removal of pyrophosphate from the highly mutagenic oxidized nucleoside triphosphate 8-oxo-2'-deoxyguanosine-5'-triphosphate (8-oxo-dGTP). Interest in MTH1 as a potential new target for cancer therapy surged due to reports of MTH1 inhibitors causing DNA damage and inducing cell death in cancer cells. However, questions have been raised about MTH1 target validation. One critical piece of information that is currently lacking is a quantitative understanding of the levels of 8-oxo-dGTP in cancer cells and the effect of MTH1 inhibition on them. In this study, we developed a sensitive and selective method to simultaneously measure the intracellular concentrations of 8-oxo guanosine nucleotides and their unmodified counterparts using liquid chromatography coupled to tandem mass spectrometry (LC-MS/ MS). An ion-pairing reversed phase liquid chromatography method using dimethylhexylamine was employed to quantify the highly polar analytes. The method was validated fit for purpose using a combination of authentic standards and cell samples. The intracellular oxo-nucleotide concentrations in human bone osteosarcoma cell line U2OS were determined using the method. We observed very low levels of 8-oxo nucleotides in untreated and hydrogen peroxide treated cells and MTH1 knockdown in these cells had minimal if any effect on 8-oxo nucleotide levels. This method will allow for a more comprehensive understanding of oxidized nucleotide detoxification pathways and MTH1 as a target for cancer therapy.