This paper describes the quantitative method of determination of chosen substances from resorcylic acid lactones group: zeranol, taleranol, α-zearalenol, β-zearalenol, and zearalanone in bovine muscle tissue. The presented method is based on double diethyl ether liquid-liquid extraction (LLE), solid phase extraction (SPE) clean up, and gas chromatography mass spectrometry (GC-MS) analysis. The residues were derivatised with a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide, ammonium iodide, and DL-dithiothreitol (1,000:2:5, v/w/w). The GC-MS apparatus was operated in positive electron ionisation mode. The method was validated according to the European Union performance criteria pointed in Decision Commission 2002/657/EC. The average recoveries of all analytes at 1 μg kg-1 level were located between 83.7% and 94.5% values with the coefficients of variation values <25%. The decision limits (CCα) and detection capabilities (CCβ) for all analytes ranged from 0.58 to 0.82 μg kg-1 and from 0.64 to 0.94 μg kg-1, respectively. The procedure has been accredited and is used as a screening and confirmatory method in control of hormone residues in animal tissues.