A high performance liquid chromatography with evaporative light scattering detector was developed for the determination of main components in polymyxin B and polymyxin E crude drugs.Analytes were separated on a Phenomenex Kinetex Biphenyl column(50 mm×4.6 mm id,5 μm) by gradient elution with methanol and 20 mmol/L ammonium acetate aqueous solution containing 0.6% formic acid as mobile phases at a flow rate of 0.5 mL/min.The parameters for evaporative light scattering detector were as follows:drift tube temperature:55 ℃,carrier gas pressure:20 psi,and the gain:250.Results showed that there were good linear relationships for the analytes in polymyxin B and polymyxin E in the concentration range of 10-200 μg/mL with their detection limits of 0.8 μg/mL and 1.2 μg/mL,and their quantitation limits of 2.6 μg/mL and 4.0 μg/mL,respectively.The intra batch recoveries for polymyxin B and polymyxin E were in the ranges of 100%-104% and 101%-103%,with intra batch relative standard deviations of 0.90%-2.4% and 0.90%-2.6%,respectively.The inter batch recoveries were in the ranges of 101%-103% and 101%-102%,with inter-batch relative standard deviations of 1.1%-1.9% and 1.3%-1.7%,respectively.Compared with microbiological detection method,the developed method is simpler,more precise and more accurate in the detection of analytes in crude drugs,and could be applied to the quality control of polymyxin B and polymyxin E crude drugs.