A method of ultrahigh performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS） was developed for the simultaneous determination of five alkaloids,including peimisine,sipeimine,peiminine,peimine and sipeimine 3β-D-glucoside in Fritillaria.Samples were extracted with alkaline methanol-chloroform,and separated on an Acquity UPLC BEH C18 column using acetonitrile solution containing 0.1% formic acid as mobile phase by gradient elution.The target compounds were determined by UPLC-MS/MS under multiple reaction monitoring（MRM） mode,and then quantified by external standard method.The calibration curves showed good linearities in the range of 0.000 5-0.05 mg/L with correlation coefficients（r） greater than 0.999.Recoveries for the five alkaloids at three spiked levels(1.0,2.5,5.0 mg/kg) ranged from 78.6% to 93.1%,with relative standard deviations（RSD） not more than 8.6%.The limits of detection（LOD） and limits of quantitation（LOQ） were 0.03 mg/kg and 0.1 mg/kg,respectively.The established method was simple,sensitive,accurate and reliable,and could be applied in the simultaneous determination of the five alkaloids in Fritillaria.