Endocrine disrupting chemicals (EDCs) can negatively affect the reproductive system, as evidenced by studies on animal models and human cell lines (Ratan S. et al., J. Endocrinol, 233(3), 2017). Exposure to such chemicals may cause detrimental effects on female reproductive health, increasing the need to improve regulatory reproductive toxicity assessment (van Duursen M.  et  al., Int. J. Mol. Sci, 21(3215), 2020). Broadening of the methods currently adopted, for example to include assessment of effects on oocyte maturation and developmental competence acquisition, is needed. Here, we present an assay identifying EDCs eliciting reproductive toxicity based on a bovine model of in vitro oocyte maturation and embryo production. To show the applicability of the assay, the known human-relevant endocrine disruptor ketoconazole (KTZ; a CYP450 inhibitor) was used. Endpoints explored include nuclear maturation, cumulus cell expansion, steroidogenesis and blastocyst rate. Immature cumulus-oocyte complexes (COC) were isolated from ovaries excised from slaughterhouse cows post-mortem. The COCs were in vitro matured for 24h in defined culture media, as previously described (Brinkhof B.  et  al., BMC Gen., 16(1), 2015), and supplemented with KTZ (10-8 M, 10-7 M, 10-6 M; Sigma-Aldrich, Missouri, USA) diluted in DMSO (vehicle; Sigma-Aldrich, Missouri, USA). Oocytes were stained with DAPI to evaluate nuclear maturation based on the presence of an MII plate and a polar body. KTZ- and vehicle-treated oocytes had comparable nuclear maturation rates (ascending KTZ M; 81%±3 ,76%±3, 65%±15 vs 84±1 vehicle-treated, n=486). Similarly, KTZ did not affect cumulus cell expansion (n=725); measured as fold-increase of the projected COC surface and calculated from images acquired pre- and post-IVM. To further investigate cumulus cell function, steroid hormone secretion into the media was quantified by LC/MS. Progesterone, estrone, and 17β-estradiol were present in levels above the detection limit. Progesterone secretion by COCs exposed to 10-6 M KTZ was reduced by 88% (733±168 pg/ml vs 6326±1169 pg/ml, one-way ANOVA, p < 0.001, n=1050), not observed at lower KTZ doses. To determine effects on developmental competence, exposed COCs were used for IVF and zygotes were cultured in vitro (IVC) for 8 days without KTZ. Oocyte exposure to 10-8 M KTZ resulted in a reduction of the D8 blastocyst rate (21%±5 vs 31%±3, n=1246, one-way ANOVA, p < 0.05), not observed at higher KTZ doses. To assess impact on early embryo development, zygotes produced from non-exposed oocytes were exposed to KTZ in IVC. No effect on D8 blastocyst rate was observed at any KTZ dose (n=991). At least three biological replicates were performed for all endpoints. In conclusion, exposure of COCs to KTZ impacted developmental competence and steroidogenesis. Oocytes were more sensitive to KTZ than cumulus cells, while embryos were not sensitive to KTZ. In the future, we will be expanding this assay to include more endpoints (e.g. cytoplasmic maturation, blastocyst quality) and verify the validity of the model for a range of EDCs with distinct action.