A high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) based method has been developed and validated to confirm the presence of the synthetic growth promoter 17a-methyltestosterone (MT) in bovine serum samples. After a single-step extraction with acetonitrile, the enhanced determination of methyltestosterone was achieved by detecting its oxime derivative. In accordance with the criteria defined in Commission Decision 2002/657/EC and using deuterated testosterone (T-d3)as an internal standard, the method has been validated in terms of decision limit (0.05 ng/mL), detection capability (0.09 ng/mL),
repeatability CV (7.04%), within-laboratory reproducibility CV (11.00%), and accuracy (87.00%).