To study the alkaloids separation and purification method from leaves of kusnezoff monkshood root using Preparative High Performance Liquid Chromatography(PHPLC).Methods:benzene extraction method was used to obtain benzene extract from leaves of kusnezoff monkshood root.PHPLC was used to separate ingredients of benzene extract from leaves of kusnezoff monkshood root.Thin layer chromatography(TLC)and High Performance Liquid Chromatography(HPLC)were used to analyze the separated ingredients and determine the target peak.PHPLC was used to purify the target peak,the peaks were collected,the separated products were analyzed by TCL and HPLC to verify the separation effect.Results:A total of 12 Peaks were collected using PHPLC separation,designated as Peak 1~Peak 12.TLC and HPLC analysis showed that Peak 5~Peak 10 were enriched in alkaloids.Peak 5 was selected as an example to receive further PHPLC separation.A total of 12 peaks were seen,defined as Peaks 5-1~Peak 5-12.TLC and HPLC illustrated that Peak 5-4 contained most of alkaloids in Peak 5,which was the target separation product.Conclusion:Chromatographic conditions established by PHPLC primary separation and secondary separation can provide basis for the large-scale extraction of alkaloids from leaves of kusnezoff monkshood root.