Abstract: To establish an UPLC-MS/MS method for simultaneous determination the contents of yunaconitine,crassicauline A and talatisamine in the five roots of Yunnan Aconitum vilmorinianum Kom,the analytical method has been developed on Agilent C18 column and the mobile phase containing ACN and 0.1% FA in water which was running in gradient elution way.The mass spectrometric acquisition was carried out by electospray ionization in positive mode with multiple reaction monitoring method,multiple characteristic ion pairs and the external standard method were opted to qualitative and quantitative analysis for yunaconitine,crassicauline A and talatisamine respectively.The linear ranges were 1—100ng·mL-1 for yunaconitine,crassicauline A and talatisamine (correlation coefficient were over 0.9990).The detection limits were 0.01,0.01ng·mL-1 and 0.02ng·mL-1 respectively;The quantitation limits were 0.03,0.04ng·mL-1 and 0.07ng·mL-1.The accuracies were all higher than 90%,The intra- and inter-day precisions were all less than 5%.Yunaconitine,crassicauline A and talatisamine were all detected in five roots of Yunnan Aconitum vilmorinianum Kom.It all contains yunaconitine,crassicauline A and talatisamine in five different roots of Yunnan kusnezoff monkshood,but the different roots contain different amount of yunaconitine,crassicauline A and talatisamine.