The estrogenic mycotoxin zearalenone (ZEN) can undergo hepatic reductive metabolism to form the estrogenic α and β isomers of zearalenol. ZEN also undergoes cytochrome P450 monooxygenase (P450)-mediated oxidative metabolism to form monohydroxylated products, but until now nothing is known about the estrogenic potency of these metabolites. This study aimed at investigating the metabolism of ZEN by different P450 isoforms and to determine the estrogen receptor α (ERα) affinities of the in vitro P450-generated ZEN metabolites in an online high-resolution screening (HRS) setup. Human liver microsomes (HLM), recombinant P450s, and mutants of the bacterial P450 BM3 were used to investigate the oxidative metabolism of ZEN. It was shown that mutants of the bacterial P450 BM3 could be used to produce the human relevant 13- and 15-OH-ZEN catechol metabolites at such levels that their ERα affinity could be determined in an HRS setup, which was not possible with HLM. It was demonstrated that P450-mediated hydroxylation at the 13 and 15 positions of ZEN resulted in a loss of ERα affinity. The approach presented here can be used for the elucidation of the metabolism of other endocrine disrupting compounds and xenobiotics to get clear pictures of the total effects of these compounds and their metabolites.