A specific and sensitive multi-method based on liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization (LC-APCI-MS/MS) has been developed for the determination of 20 anabolic steroids in muscle tissue (diethylstilbestrol, beta-estradiol, ethynylestradiol, alpha/beta-boldenone, alpha/beta-nortestosterone, methyltestosterone, beta-trenbolone, triamcinolone acetonide, dexamethasone, flumethasone, alpha/beta-zearalenol, alpha/beta-zearalanol, zearalenone, melengestrol acetate, megestrol acetate and medroxyprogesterone acetate). The procedure involved hydrolysis, extraction with tert-butyl methyl ether, defattening and final clean-up with solid phase extraction (SPE) on Oasis HLB and Amino cartridges. The analytes were analyzed by reversed-phase LC-MS/MS, in positive and negative multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for the unambiguous confirmation of the hormones. The method was validated at the validation level of 0.5ng/g. The accuracy and precision of the method were satisfactory. The decision limits CCalpha ranged from 0.03 to 0.14ng/g while the detection capabilities CCbeta ranged from 0.05 to 0.24ng/g. The developed method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in muscle tissue and can be used for residue control programs.