Abstract: A liquid chromatography-mass spectrometry method was developed for the qualitative analysis of eleven β_2-agonists in human urine. This method was successfully applied to the analysis of the urine samples excreted by some healthy subjects over different periods after oral administration of single dose of β_2-agonists. Urine samples were hydrolyzed with β-glucuronidase followed by a solid-phase extraction procedure using Bond Elut-Certify cartridges. The analytes were separated on Agilent Zorbax SB-C_ 18column using a gradient elution of (A) 0.01 mol/L ammonium formate buffer (pH 3.5) and (B) acetonitrile. Elution started with 95% A, followed by a linear gradient to 45% A in 8 min, and decreased to 10% A in another 5 min, and then held 6 min. An electrospray ionization (ESI) source was operated in positive ion mode. The detection limits of 11 β_2-agonists were in the range of 0.1-60.0 μg/L.