This paper developed a universal detection method by high-performance liquid chromatography-tandem mass spectrometry to detect three typical clenbuterols, CLB, SAL, and RAC, and to investigate the metabolism of β-agonists in vivo. The parent ions and daughter ions of the three β-receptor agonist standards and the residues in the muscle, liver, and blood samples of rats were obtained by Total Ions Scan mode. The metabolites produced in different tissues at a specific time were qualitatively and quantitatively analyzed, and the corresponding metabolic pathways were inferred. The results showed that the three β-receptor agonists mainly existed in the form of prototype drugs in rats, with a small amount of clenbuterol methyl compound and albuterol methyl compound. There were significant differences in residual metabolism between different tissues of the same species. In addition, different β-receptor agonists have different absorption and utilization rates in rats.