Abstract: A method for fast determination of 1-hydroxypyrene (1-OHP) in human urinary by microwave assisted enzymatic hydrolysis-ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed in this paper.The results of enzymatic hydrolysis compared with that of alkaline hydrolysis,microwave assisted enzymatic hydrolysis compared with that of water bath enzymatic hydrolysis,and the liquid-liquid extraction compared with that of solid phase extraction were discussed,respectively.The preparation of standard solutions by blank urine and pure solvent were studied to reduce the matrix effect. Finally,the microwave assisted enzymatic hydrolysis and solid phase extraction were selected and the blank urine was used to prepare the standard solutions.The standard curve equation of the method was y=1.241 07x-0.130 042.The correlation coefficient was 0.9972.The recovery was 88.6%—97.1%.The relative standard deviation was 2.7% to 12.6%.The intra-day precision was 6.1%.The inter-day precision was 6.2%.The detection limit was 0.08 ng /mL.The limit of quantification was 0.28 ng /mL.Compared with other existing methods,the established method in this paper for determination of urinary 1-hydroxypyrene was more simple and accurate.Its efficiency,reproducibility and sensitivity were also satisfying.