Introduction: The use of growth promoters in animal husbandry to increase weight gain and efficiency of feed conversion into muscle has been banned in the European Union since 1988, and under Directive 96/23/EC, surveillance for anabolic steroid hormones is obligatory. The hormones present in animal tissues may be of endogenous origin or may result from illegal administration. Steps have been taken to determine selected steroids in the form of esters in the alternative matrix of animal hair. Their detection in biological material is direct proof of the illegal use of anabolics. Material and Methods: The procedure for the determination of steroid esters in animal hair, based on digestion, extraction, purification, and liquid chromatography with tandem mass spectrometry was validated under the current regulations. In total, 348 samples of animal hair were examined using this method. Results: Good recoveries and precision values (RSD) were obtained during validation. Decision limits (CCα) and detection capabilities (CCβ) were in the ranges of 2.57–4.18 μg kg−1 and 4.38–7.12 μg kg−1, respectively. The method met the criteria for confirmation techniques with respect to Commission Decision 2002/657/EC. Conclusion: Testing for steroid esters in animal hair was introduced into the National Residue Control Programme in 2017. Steroid esters were not found in any hair samples above the CCα, which indicates that illegal use of anabolics was not confirmed.