Screening of pharmaceutical, chemical and environmental compounds for their effects on reproductive health relies on in vivo studies. More robust and efficient methods to assess these effects are needed. Herein we adapted and validated an organotypic in vitro follicle growth (IVFG) assay to determine the impact of compounds on markers of ovarian function. We isolated mammalian follicles and cultured them in the presence of compounds with: (1) known fertotoxicity (i.e. toxicity to the reproductive system; cyclophosphamide and cisplatin); (2) no known fertotoxicity (nalbuphine); and (3) unknown fertotoxicity (Corexit EC 9500 A; CE, Nalco, Chicago, IL, USA). For each compound, we assayed follicle growth, hormone production and the ability of follicle-enclosed oocytes to resume meiosis and produce a mature egg. Cyclophosphamide and cisplatin caused dose-dependent disruption of follicle dynamics, whereas nalbuphine did not. The reproductive toxicity of CE, an oil dispersant used heavily during the 2010 Deepwater Horizon oil spill, has never been examined in a mammalian system. In the present study, CE compromised follicle morphology and functional parameters. Our findings demonstrate that this IVFG assay system can be used to distinguish fertotoxic from non-toxic compounds, providing an in vitro tool to assess the effects of chemical compounds on reproductive function and health.