Abstract A liquid chromatography–tandem mass spectrometric (LC–MS/MS) multi-method has been developed for the determination of 15 anabolic steroids in bovine urine (diethylstilbestrol, dienestrol, hexestrol, β-estradiol, ethynylestradiol, α/β-boldenone, α-nortestosterone, α/β-zearalenol, α/β-zaeralanol, zearalenone, stanozolol and 16β-OH-stanozolol). The procedure involved enzymatic hydrolysis, extraction with tert-butyl methyl ether, a washing step with hexane and final clean-up with SPE with Oasis HLB and Amino cartridges. The analytes were quantified by liquid chromatography coupled to a tandem mass spectrometer (LC-TSQ Quantum AM) operating in both positive and negative atmospheric pressure chemical ionisation (APCI). Data acquisition was performed in multiple reaction monitoring (MRM) mode quantifying two diagnostic product ions from a chosen precursor. The method was validated according to the Commission Decision 2002/657/EC, for the detection and confirmation of residues in products of animal origin. The method specificity, sensitivity, accuracy and precision were evaluated. The decision limits CCα ranged from 0.06 to 0.26 ng/ml and the detection capabilities CCβ ranged from 0.11 to 0.49 ng/ml. The developed method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in bovine urine and can be used for residue control programs.