A variety of natural and synthetic environmental substances have been shown to disrupt vertebrate reproduction through mimicking or modifying the regulation of the endocrine system. Tests to screen for any such chemicals that directly interact with the steroid hormone receptors are widely available; however, few tests have been developed to identify chemicals that affect endocrine function through non-receptor mediated mechanisms. The aim of this study was, therefore, to develop an assay for the identification of substances that disrupt the activity of enzymes involved in the sex steroid biosynthesis cascade, in particular the aromatase enzyme, CYP19, that catalyses the final conversion of androgens to estrogens. A gonad ex vivo assay was developed using gonad explants harvested from juvenile brown trout and cultured in a modified Leibovitz medium. Effects on sex steroid biosynthesis were quantified through measurement of 17β-estradiol (E2) and testosterone (T) concentrations in the medium after 2 days incubation. Exposure of ovary explants to 100 ng/mL 1,4,6-androstatriene-3,17-dione (ATD), a potent pharmaceutical aromatase inhibitor, reduced E2 concentrations and elevated T concentrations confirming that CYP19 activity could be inhibited in the assay. Exposure of ovary explants to 250 ng/mL prochloraz, an imidazole fungicide, also reduced E2 concentrations but did not affect T levels, consistent with reports that in addition to inhibiting CYP19 activity, prochloraz also inhibits enzymes in the steroidogenic pathway upstream of the CYP19 enzyme. Exposure to a third chemical, tributyltin (TBT), did not affect T or E2 concentrations, further supporting previous evidence that the CYP19 modulating effects of this chemical are not mediated through direct inhibition of CYP19 activity. These results demonstrate that the gonad ex vivo assay developed here can be successfully used to identify substances that disrupt sex steroid biosynthesis and further that it has the potential to inform on their specific mode of action.