Abstract: In order to evaluate the exposure to PAHs in environmental tobacco smoke, the influences of solid phase extraction column, chromatographic column and mass spectrometry conditions on analytical results were investigated, a method with SPE-HPLC/MS/MS was established to determine the biomarkers of 1-OHNap, 2-OHNap, 1-OHPyr, 6-OHChr, 3-OHFlu, 1-OHPhe, 2-OHPhe, 3-OHPhe, 4-OHPhe, 9-OHPhe and 2-OHFlu in human urine. Using 1-OHNap-d₈, 3-OHPhe-d₉ and 1-OHPyr-d₉ as internal standards, the samples were hydrolyzed by β-glucosidase/aryl sulfatase, extracted by Bond Elut C₁₈ cartridge, separated by Eclipse PAH column, determined by mass spectrometry under multi-reaction monitoring mode(MRM),the OH metabolites of PAHs (OHPAHs) in urine were detected. The results showed that the limits of quantification (LOQ) of OHPAHs varied from 6 to 130 pg/mL. The detectable concentration of OHPAHs showed good linear relationship with r from 0.9991 to 1.0000, and the recoveries ranged from 72.45% to 124.50%. The intra-day and inter-day precisions were in the range of 1.49% to 7.35% and 2.90% to 12.00%, respectively. This method is suitable for simultaneously determining the biomarkers of PAHs in human urine.