Abstract: A high performance liquid chromatography-electrospray ionization tandem mass spectrometry method was developed for a simultaneous detection of 40 prohibited substances,coveredβ-blockers and narcotics,some stimulants andβ2-agonists which are included in the prohibited list issued by the World Anti-Doping Agency(WADA).After the pretreatment with acid hydrolysis,extraction and enrichment procedures,the urine sample was injected to the LCMS/MS.The limits of detection were between 0.1~10ng/mL according to different substances.The spiked recoveries at low concentration,WADA Minimum Required Performance Levels(MRPL)and high concentration were within the range of 1.6% to 118.6%.This method was a simple,fast,effective and reliable solvent to our laboratory and now has been successfully applied to the routine analysis and the External Quality Assessment Scheme set out by WADA.