Current veterinary residue analysis mainly focuses on the monitoring of residues of the administered parent compound. However, it is possible that larger amounts of metabolites are excreted and that they can have a prolonged excretion period. In order to unravel specific metabolic steps and to identify possible biological markers, two in vitro liver models were used, i.e. monolayer cultures of isolated hepatocytes and liver microsomes, both prepared from liver tissue of cattle. Chostebol, boldenone, norethandrolone (NE) and ethylestrenol (EES) were used as model substrates. Results show that the metabolic profiles derived from in vitro experiments are predictive for the in vivo metabolic pathways of the steroids evaluated in this study. By means of this strategy, it is possible to identify 17 alpha-ethyl-5 beta-estrane-3 alpha,17 beta-diol (EED) as a common biological marker for NE and EES. By in vivo experiments it was shown that EED is particularly important for the detection of the abuse of NE or EES because of its high excretion levels and its prolonged presence as compared with the parent compounds or any other metabolite.