To establish an ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analytical methods and confirmatory method for positive samples to determine seven β₂-receptor agonists including mabuterol, cimaterol, ractompamine, clenbuterol, salbutamol, terbutaline and isoxsuprine in animal derived samples. Methods The animal derived samples were enzymatic hydrolyzed and extracted with ammonium acetate buffer solution. The supernatant was treated by cation exchange column solid phase extraction (SPE), followed by nitrogen concentrating and analyzed by UHPLC-MS/MS. The suspicious positive sample was further confirmed by specific scanning mode of QTrap tandem mass spectrometry. This study also investigated the matrix effects of seven β₂-receptor agonists.Results The results showed that there was a strong matrix effects of the method. The correlation coefficients were good in the concentration range of 0.25-50 ng/g. The limit of detection was 0.1-0.5 ng/g. The average recovery for β₂-receptor agonists was in the range of 86.6%-108.7%.Conclusion The internal standard quantitative method could be used to reduce the quantitative error from the matrix effect. This method could be used for determination of β₂-receptor agonists in animal derived samples with a low detection limit, good accuracy and fast. The positive samples could be further confirmed.