Abstract: Monocrotaline was separated from sample of crotalaria sessiliflora by ultrasonic extraction with methanol.Influential factors of extraction,including the extraction solvents,time and temperature of extraction as well as the ratio of mass of sample to volume of extraction solvent,were studied and optimized by the method of orthogonal design with L9(34) orthogonal layout.In the HPLC determination,UltimateTM C18 (4.6 mm×250 mm,5 μm) column was used as stationary phase and a mixture of methanol and triethylamine-water (0.5+99.5,pH 7.0) solution,mixed in the ratio of 30 to 70,was used as mobile phase.Linear relationship between values of peak area and concentration of monocrotaline was obtained in the range of 1.0-800 mg·L-1.Detection limit (3S/N) found for the method was 0.1 mg·L-1.Recovery was tested by standard addition method and values of recovery found were ranged from 97.8% to 102.3%.