In the determination of nine β-agonists including salbutamol, terbutaline, cimaterol, fenoterol, clorprenaline, ractopamine, tulobuterol, clenbuterol and penbuterol in porcine, bovine, lamb and chicken muscle, liver and urine samples with LC-MS/MS, calibration curves prepared in solvent (SC) were compared with those prepared in each matrix (MC) for all analytes. Significant differences (P < 0.05) between SC and each MC for most analytes indicated the existence of matrix effects and the necessity of using MC for quantitation to compensate MEs. Then MC in each muscle, liver and urine sample was compared with the select potential representative matrix, followed by validating the recoveries of nine analytes calculated through the MC in the potential representative matrix and MC in their corresponding matrices, respectively. The results suggested that porcine muscle could be selected as a representative matrix to calibrate β-agonist residues in bovine, lamb and chicken muscle samples.