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8-Hydroxydeoxyguanosine (8-OHdG) is a widely-used biomarker of oxidative DNA damages. 8-OHdG in peripheral blood leukocyte is associated with mutation and cancer risk. The level of 8-OHdG in peripheral blood leukocytes can indicate a long-term response to oxidative stress rather than that in urine. Accurate identification and quantification of leukocyte 8-OHdG are essential for understanding its mechanism of formation, repair, and biological consequences. In this study, a fast and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated to detect 8-OHdG in human peripheral leukocyte. DNA in blood samples were extracted and digested, then subjected onto UHPLC–MS/MS using an isocratic elution on a Zorbax Eclipse Plus C18 column (2.1 × 100 mm, 1.8 μm). Multiple reaction monitoring (MRM) mode was adopted by using [ 15 N 5 ]-8-OHdG as an internal standard. The assay was linear over the concentration range of 1.0–100 nM with R 2 = 0.999. The accuracy for spiked samples was 90.9%–94.8%, and the intra-day precision was within 3.7%. The limit of detection (LOD) is 0.30 nM and limit of quantification (LOQ) is 1.0 nM with 5 μl of sample injection. By the analysis of human leukocyte 8-OHdG (n = 121) using the developed UHPLC–MS/MS method, it demonstrates that the level of leukocyte 8-OHdG of the cancer patients group (n = 46) is significantly higher than that of the health control (n = 75). [ABSTRACT FROM AUTHOR]