Abstract: A method for the determination of wilfortrine in urine by HPLC-MS was proposed. Wilfortrine in sample was separated, enriched and purified on the Waters Oasis MCX SPE column. The eluate was used for HPLC-MS determination, in which the Zorbax Eclipse SB C18 column (4.6 mm×250 mm, 5 μm) was used as chromatographic column, and a mixture of acetate buffer solution and acetonitrile (40+60) was used as mobile phase. Atmospheric pressure chemical ionization source with positive ionization mode as well as selected ion monitoring mode was used in the detection. Ion used for quantification was m/z 874.4. Linearity range was found between 0.2-50.0 μg·L-1, and values of detection limit (3S/N) and lower limit of determination (10S/N) found were 0.07 μg·L-1 and 0.2 μg·L-1 respectively. Values of recovery were ranged from 86.0% to 92.0%. Values of RSD′s (intra-day, n=6) and RSD′s (inter-day, n=15) were less than 7.5% and 10.5%, respectively.