Abstract: In order to accurately determine levels of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolites in mouse blood and the metabolic pathways of NNK in mice, a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for simultaneous determination of NNK and its seven metabolites including 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanol(NNAL), NNK-N-oxide, NNAL-N-oxide, 4-hydroxy-1-(3-pyridyl)-1-butanone(HPB), 1-(3-pyridyl)-1,4-diol(PBD), 1- (3-pyridyl)-1-butanone-4-carboxylic acid and 1-(3-pyridyl)-1-butanol-4-carboxylic acid in the blood of C57BL/6 mice was established by Q Exactive Orbitrap High Resolution Mass Spectrometer. With NNK-d₄, NNAL-d₃, and HPB-d₄ as internal standards, after protein precipitation with acetonitrile, the serum samples of C57BL/6 mice were separated by a HILIC column and determined by a mass spectrometer under full scan/data dependent MS² mode. The results showed that the limits of quantification of the target analytes ranged from 0.042 to 0.263 ng/mL with correlation coefficients of 0.999 1 to 1.000 0, and the recoveries ranged from 89.2% to 116.5% with the intra-day precision of 1.54% to 8.06%. The developed method features good selectivity, high efficiency and large linear range, and is suitable for the simultaneous determination of NNK and its seven metabolites in mouse blood.