Abstract: A rapid high-performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD) method for determination of 24-epibrassinolide has been established. The optimized ELSD conditions were as follows: drift tube temperature is 50 ℃, gain value is 10 and the auxiliary gas pressure is 350 kPa. The determination was carried out on a Eclipse XDB C18 column (250 mm×4.6 mm, 5 μm) with the mobile phase consisted of acetonitrile and water (V/V=55:45), column temperature set at 30 ℃ and flow rate of 1.0 mL/min. Complete separation of the main composition and impurities with similar chemical structures was achieved in 10.0 min under optimized conditions. The developed method was fully validated. The results showed that the regression of logarithmic value of peak area on the logarithmic value of the mass injected fitted a 1inear relationship well (r=0.999 3) at the concentration levels ranging from 43.8-700.7 mg/L. The relative standard deviations (RSD, n=7) was 0.75% and the mean recovery was 99.7%. The optimized and validated method is efficient, accurate and easy to use, which provides reliable method for determination and quality control of 24-epibrassinolide and other brassinosteroids.