To develop an analytical method for the determination of 26 β-agonists in animal liver tissue based on ultra pressure liquid chromatography tandem mass spectrometry (UPLC-MS/MS).Methods Pig liver and duck liver were extracted using trichloracetic acid solution. Then the extract were purified and enriched by Oasis MCX solid phase extraction cartridges. Target compounds were separated on a Waters ACQUITY UPLCTM BEH C₁₈ column with gradient elution using methanol and water containing 0.1% formic acid. Quantification was performed with matrix-fortified standard calibration.Results Average recoveries of 26 β-agonists spiked at three concentrations ranged from 76.4% to 115.4% with relative standard deviation lower than 15.0%. The limits of quantitation (LOQs) for the method ranged from 0.1 to 0.7 μg/kg.Conclusion Due to its high sensitivity and good reproducibility, the method is suitable for the determination of target agnoists in animal liver tissue.