Abstract: To simplify sample pretreatment and reduce interference caused by matrix components when analyzing hydroxylated polycyclic aromatic hydrocarbons (OHPAHs), which are recognized biomarkers of polycyclic aromatic hydrocarbons (PAHs) for cigarette smokers, a new method for simultaneously determining 10 OHPAHs in human urines was developed based on two-dimensional liquid chromatography-tandem mass spectrometry system (2DLC-MS/MS). The urine samples were directly analyzed after enzymatic hydrolysis of PAHs. The analytes were separated on a C₁₈ column with water-methanol as the mobile phases in the first chromatograph separation, cut to the trap column by online dilution, and then reserved and enriched on the column. Finally the analytes were separated by gradient elution on the PAH column with water-acetonitrile as the mobile phases in the second chromatograph. MS/MS analysis was performed in multiple reaction monitoring (MRM) mode. The results showed that: 1) Baseline separation was realized for all 10 target OHPAH compounds. 2) The detection limits of the method for OHPAHs were 0.001-0.021 ng/mL and quantification limits were 0.005-0.070 ng/mL. The spiked recoveries ranged from 87.9% to 129.1% with intra- and inter-RSDs less than 5%. 3) The method was successfully applied to the analysis of the urines from 6 smokers and 6 non-smokers. This method achieved accurate quantitation of 5 hydroxyphenanthrene isomers with simple sample pretreatment and highly automated procedures, thus providing a more efficient and accurate technique for the determination of PAHs in tobacco smokers.