Abstract: A reliable method for determination of active ingredients in technical zhongshengmycin material was established using high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS).The active ingredients in technique material were extracted by the ultrasound-assisted method,and then the aqueous extract was cleaned up by centrifugation and filtration.After separated by reverse-phase ion-pair chromatography,the characteristic MS/MS ions were detected by mass spectrometer and quantified with external standard method.A good linear relationship between response and concentration was obtained in the range of 15.63-500 μg/mL,and its correlation coefficient was above 0.990 5.The relative standard derivations for different ingredients were in the range of 0.33% -1.96%.The recovery rates for different ingredients were 98.2% -101.1% in the spiking levels of 0.1,0.5 and 1 μg/mg.The contents of streptothricins D,B,C,F,A and E in the tested technical material were 297.65,247.77,285.64,115.92,15.63 and 19.60 μg/g,respectively.The method could be used to qualitative and quantitative analysis of active ingredients in technical zhongshengmycin.