Abstract: A method based on liquid chromatography with tandem mass spectrometry(HPLC-MS/MS) was developed for the simultaneous determination of 3-hydroxypropyl mercapturic acid(3-HPMA) and 3-hydroxy-1-methyl-propylmercapturic acid(3-HMPMA), the mercapturic acid metabolites of acrolein and crotonaldehyde in human urine.Using 3-HPMA-D₃ and 3-HMPMA-D₃ as internal standards, the urine samples were cleaned up by Isolute ENV+ solid phase extraction(SPE) cartridge and separated by an Angilent Extend C₁₈ column, and then 3-HPMA and 3-HMPMA in the samples were detected by the tandem mass detector under multi-reaction monitoring scan mode.The contents of 3-HPMA and 3-HMPMA in urine samples from eight smokers and eight nonsmokers were determined, the results showed that:1) the recoveries of 3-HPMA and 3-HMPMA ranged from 91.11% to 114.66% with the relative standard deviations(RSDs) of 2.41% to 11.22%, and the limit of detection was of 1.0 and 1.3 ng/mL, respectively;2) the contents of 3-HPMA and 3-HMPMA in urine samples from eight smokers ranged from 943.05 to 6303.54 and from 1061.73 to 6373.74 ng/mL, and from eight nonsmokers ranged from 14.13 to 640.93 and from 23.36 to 119.40 ng/mL, respectively.