Clostebol acetate (4-chloro-testosterone acetate) is an anabolic steroid used for fattening purposes in cattle breeding. To safeguard public health, its use has been prohibited by the European Commission since 1986. Screening for its urinary metabolites is therefore an important tool for the control of possible violations. Because those metabolites appear conjugated to glucuronic acid or sulfate, deconjugation prior to analysis is necessary. This work describes the variability in results seen with the use of various commercial preparations of Helix pomatia (SHP) for enzymatic hydrolysis of the conjugates. A simultaneous oxidative side reaction was observed, converting metabolites with a 3-OH-4-ene structure into a 3-oxo-4-ene structure. This was not observed when samples were incubated without enzyme or in the presence of heat-inactivated SHP. GC–MS analysis revealed oxidation of some metabolites of clostebol acetate, 4-chloro-4-androsten-3a-ol-17-one and 4-chloro-4-androsten-3a,17b-diol, changing them into other metabolites, 4-chloro-4-androsten-3,17-dione and clostebol (4-chloro-testosterone), respectively. Based on the difference in cross-reactivities of the antibodies for these metabolites, comparative analysis in enzyme immunoassay, following enzymatic hydrolysis, confirmed this transformation. This oxidative conversion phenomenon could be of great importance when considering the choice or target analytes for screening bovine urine.