A sensitive, specific and selective multianalyte GC-MS/MS method has been developed for the determination of 11 anabolic hormones in bovine urine. After adjusting the urine pH to 4.8, the samples were spiked with deuterated internal standards and submitted to enzymatic hydrolysis with beta-glucuronidase/arylsulfatase. Hormones were eluted with methanol through a C18 solid phase cartridge and submitted to a liquid-liquid extraction. Analytes were derivatized by adding N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane and GC-MS data were obtained in the positive electron impact tandem mass mode. Under these conditions, no matrix effects were observed and limit of detection values were in the range of 0.005 ng/mL (diethylstilbestrol) to 0.38 ng/mL (17alpha-methyltestosterone and 17alpha-ethynylestradiol). Recoveries from 81% (alpha-zeranol) to 149% (17alpha-methyltestosterone) were found under the selected conditions. These results were better than those found using heptafluorobutyric anhydride (HFBA) as derivative reagent and those measured in full scan and selective ion monitoring modes.