Abstract: 15 mg of salmeterol is administrated to 10 rats, which weighs about 25 g each. The urine of the rats is extracted by solid-phase column and analyzed by liquid chromatography-APCI-mass spectrometry. Parent drug and four metabolites are identified by comparing total ion chromatograms of the blank with the metabolic urine as well as their mass spectra. Its main metabolic pathways are the hydroxylation and/or methoxylation from the experiments.