Objective: A method for the determination of acetochlor and its metabolites in urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established. Methods: After cleaned-up by a HLB extraction cartridges, the urine was eluted with 1% acetic acid acetonitrile solution. The target compounds were separated by ACQUITY UPLC®HSS T3 Column (2.1 mm×100 mm×1.8 μm) by using 1% formic acid solution and acetonitrile as mobile phase with gradient elution program, and analyzed in positive electrospray ionization mode by liquid chromatography tandem mass spectrometry. Results: All the target compounds showed good linear relationships in the range of 1-50 μg/L, and the correlation coefficients (r) were higher than 0.997. The recoveries rates at three different spiked levels for all target compounds in blank matrices were 107.6%-129.1%, and the relative standard deviations (RSD) were 1.5%-9.9% (n=6) . The limits of detection and quantitation of the method were 0.04-0.11 μg/L and 0.15-0.42 μg/L, respectively, and target substances were detected in all urine samples from occupational exposure workers to acetochlor. Conclusion: This method is suitable for rapid screening and analysis of acetochlor and metabolites in urine with the advantages of accuracy, rapidity, simplicity, high sensitivity and good specificity.