Abstract: A methodology applying HPLC-MS/MS was developed for the determination of clonidine hydrochloride and cyproheptadine hydrochloride contents in pig urine.Samples were hydrolyzed with perchloric acid, extracted with ethyl acetate-isopropanol, and purified through an MCX SPE cartridge before feeding to a Venusil MP C18 column (100 mm×2.1 mm, 3 μm).On the column, the sample solution was flushed with a 0.1% (v/v) formic acid and 5% (v/v) acetonitrile aqueous solution, as well as a 0.1% (v/v) formic acid and 95% (v/v) acetonitrile aqueous solution as the mobile phase at the flow rate of 0.3 mL·min-1 and the column temperature of 30℃.The multiple reaction monitoring mode was used for the qualitative and quantitative analyses, and the external standard method for the quantitative determination.When the concentration of clonidine hydrochloride or cyproheptadine hydrochloride was 1.0-100.0 μg·L-1, a linear correlation (r≥0.9995) existed.At the addition of 0.2, 2 and 10.0 μg·L-1 of the standard chemicals, the recovery rates were 83.5%-95.1% with an RSD (n=6) of 3.5%-8.7% and a quantity limit of 0.2 μg·L-1 (the signal to noise ratio >10).The newly developed method appeared to be precise, sensitive, accurate and simple to operate for the determination of clonidine hydrochloride and cyproheptadine hydrochloride contents in pig urine.