Abstract: The collected urine sample was successively added with internal standard and methanol, and the volume fraction of methanol in the sample medium should be controlled to be about 50%. The above mixture was prepared by self-designed device for centrifugation and filtration of the sample. HPLC-MS/MS was applied to the rapid determination of 8-hydroxydeoxyguanosine and 8-hydroxyguanosine in the filtrate. Synergi Polar-RP chromatographic column was used as stationary phase, and the mixture of 10 g·L^-1 ammonium acetate solution and methanol mixed in different ratios was used as mobile phase in gradient elution. ESI⁺ and multi-reactions monitor mode were adopted in MS/MS. ¹⁵N₅-8-hydroxydeoxyguanosine was used as internal standard. Linearity ranges of 8-hydroxydeoxyguanosine and 8-hydroxyguanosine were found in the same range of 0.2-40 μg·L^-1, with detection limits (3S/N) of 0.054, 0.092 μg·L^-1 and lower limits of determination (10S/N) of 0.18, 0.32 μg·L^-1. On the base of urine sample of a moderate smoker, test for recovery was made by standard addition method; values of intra-day recovery and inter-day recovery were in the ranges of 92.3%-96.3%, 93.4%-96.3% respectively, with intra-day RSDs (n=7) and inter-day RSDs (n=7) of determined values in the ranges of 1.6%-3.0%, 2.8%-3.4% respectively.