Abstract: Tobacoo-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen present in tobacco products and tobacco smoke. The carcinogenic mechanism of NNK is involved in the DNA damage induced by the active electrophiles generated from the metabolic activation of NNK catalyzed by cytochrome P450. It is significant to establish an in vitro metabolism model of NNK and the quantitation method for the investigation of tabacco carcinogens motabolites. In this work, the NNK metabolite of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) was quantitatively analyzed by high performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (HPLC-APCI-MS/MS). Selective reaction monitoring (SRM) was employed for determining HPB and the internal standard 3,3,4,4-D₄HPB simultaneously. The method shows good linearity within 0.2—400 nmol/L with the correlation coefficient (R₂) 0.999 9. The limit of detection (LOD) and the limit of quantitation (LOQ) are 0.025 nmol/L (S/N=3) and 0.05 nmol/L (S/N=10), respectively. The inter-day and intraday accuracy range from 96.6% to 101.8%, and the recovery is 98.1%—102.6%. The in vitro metabolism model of NNK can be used to elucidate the molecular mechanism of the metabolic activation of tobaccospecific nitrosamine. This work layed a foundation for the quantification of biomarkers for tobacco carcinogenesis.