Abstract: A method for the simultaneous determination of 36 kinds of veterinary drugs in antler velvet of cervus by UPLC-MS/MS was developed by using the pretreatment technology of dispersed solid phase extraction. Antler samples were extracted with acetonitrile and purified with 150 mg PSA, 100 mg C18 and 70 mg neutral alumina. The target analytes were separated by Acquity BEH C18 column (2.1 mm×100 mm×1.7 μm) with the gradient elution using 25 mmol/L formic acid acetonitrile and 12.5 mmol/L formic acid water as mobile phases. The analytes were detected by positive electrospray ionization with multiple reaction monitoring. The content of 36 kinds of veterinary drugs in pilose antler was quantified by external standard method. The linear relationship of 36 veterinary drugs was good in the range of 0.1-50 μg/L, the correlation coefficients were all greater than 0.993, the LOD was 0.08-1 μg/kg, and the LOQ was 0.3-3 μg/kg. 36 kinds of veterinary drugs with concentrations of 2, 5, 10 μg/kg were added to the blank sample. The average recovery was 77.8%-107.6%, and the relative standard deviation was less than 10%. The method has the advantages of simple operation, remarkable purification effect, high sensitivity, good accuracy and low detection limit, which can be used for the determination of 36 kinds of veterinary drugs in antler velvet of Cervus nippon Temminck.