Abstract: A method for the determination of euonine in human urine by LC-MS was proposed. Euonine in sample was separated, enriched and purified on Waters Oasis MCX SPE column. The SPE column was eluted with ammoniacal acetonitrile. The eluate was used for LC-MS determination. Zorbax Plus RRHD C18 column(50 mm×2.1 mm, 1.8 μm) was used as chromatographic column, and a mixture of acetate buffer solution and acetonitrile (30+70) was used as mobile phase in the separation. Atmospheric pressure chemical ionization source with positive ionization mode as well as selected ion monitoring mode was used in the detection. Quantitative ion of euonine used was m/z 806. Linear relationship between values of peak area and mass concentration of euonine was kept in the range of 0.2-50.0 μg·L-1, with lower limit of determination (10S/N) of 0.2 μg·L-1. Values of recovery were ranged from 84.5% to 94.0%. Values of intraday RSD′s (n=6) and interday RSD′s (n=5) found were less than 7% and 10%, respectively.