A new and rapid method was developed and validated for the simultaneous analysis of 15 alkaloids，including anabasine，gramine，eserine，pilocarpine，harmine，oxymatrine，thermopsine，koumine，gelsemine，tetrahydropalmine，evodiamine，sanguinarine，chelerythrine，strychnine and brucine in urine.The conditions of extraction，chromatography and mass spectrometry were optimized，and the matrix effects of the alkaloids were also examined.The detection of target compounds was performed by gas chromatography-tandem mass spectrometry in multiple-reaction monitoring(MRM) mode.The content of each alkaloid was determined by the external standard method.The linear ranges were 20-800 μg/L for eserine，harmine，koumine and strychnine，and 40-800 μg/L for other target alkaloids with correlation coefficients not less than 0.993 2.The mean recoveries were in the range of 60.0%-68.3% for anabasine，and 81.9%-114.4% for other alkaloids.The relative standard deviations(RSDs) were not more than 17.6% at three spiked concentration levels.The limits of detection(LODs) and quantitation(LOQs) were 4-20 μg/L and 10-40 μg/L，respectively.The method was rapid，sensitive and easy operation，and was suitable for the determination of trace alkaloids in urine samples of poisonous persons.