Abstract: In order to evaluate the carcinogenicity of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), an ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) method was established to accurately determine the levels of 6-O-methylguanine (6-O-MeG)in mouse liver and lung tissues. The contents of 6-O-MeG in the liver and lung of normal saline-treated group, pyrazole-treated group and 5-methoxypsoralen (5-MOP)-treated group were measured separately after NNK administration. Tissue samples were extracted by DNA extraction kits, then the extraction was acidified at 80℃, followed by addingCD₃ 6-O-MeG as the internal standard. After going through HLB solid phase column and redissolution by acetonitrile, the extraction was analyzed by UPLC-HRMS. The results showed that:1)6-O-MeG displayed good linearity in the range of 0.05-10.00 ng/mL with R² of 0.999 8, the limit of detection (LOD)of 0.011 ng/mL and the recoveries from 92.1% to 102.2%, the RSDs of intra-and inter-day were 1.10%-4.34% and 1.89%-6.58%, respectively. 2) The concentration of 6-O-MeG in the liver and lung of mice treated with pyrazole was 2.09 and 1.87 times as high as those treated with saline four hours after NNK administration; the 5-MOP-treated group was 0.32 and 0.67 times as high as the control group. The results indicated that cytochrome P450 2A5 (CYP2A5) enzyme played an important role in the activation of NNK metabolism in mice. This method features a simple sample preparation, good selectivity, high sensitivity, and it is suitable for determining 6-O-MeG in mouse tissue.