Abstract: OBJECTIVE To develop an high performance liquid chromatography with triple quadrupole mass spectrometry (HPLC-MS/MS) method to determine 3 β₂-agonists including clenbuterol, salbutamol and ractopamine in animal-derived Chinese medicine. METHODS The analysis was performed by a HPLC-MS/MS system with Agilent XDB-C₁₈(50 mm×4.6 mm, 5.0 µm) column. Multiple-reaction monitoring (MRM) was performed to identify and quantify 3 β₂-agonists, which were extracted from Asini Colla Corii and Bovis Calculus Artifactus, purified with MCX SPE columns. RESULTS three linear calibration curves were obtained with R² ≥ 0.997 1. The precision of the method were showed by RSD (n=6) ranged from 1.4% to 2.3%. The recoveries were determinated at three concentrations and ranged from 89.4% to 102.8%. The ranges of LOQ were from 0.25 µg·L^-1 to 5.50 µg·L^-1, and the RSDs of intra-day precision (n=5) and inter-day precision (n=9) were from 2.4% to 4.6% and from 1.9% to 3.7%. CONCLUSION The method is specific, simple and fast to detect the 3 β₂-agonists in animal-derived Chinese medicine.