Major limitations in understanding the direct effects of endocrine-disrupting chemicals (EDCs) and cell signaling events in ovarian cellular dynamics in mammals include a lack of proper and simple tools/techniques as well as gaps in knowledge regarding the critical window(s) of vulnerability. Identifying and validating such tools and evaluating the effects of EDCs on molecular dynamics and cellular events during the critical windows of ovarian development are very important to improve the fertility in women and preserve the future health of the developing fetuses. Therefore, we developed a fetal whole ovarian ex vivo culture model. Ex vivo ovary culture models allow varying culture parameters in a highly controlled manner and thus have the potential to allow a more thorough evaluation for reproductive toxicity studies and drug response. This chapter describes clear and thorough details for setting up and maintaining an ex vivo culture system from the rat ovaries and further analyses of mRNA and protein expressions and estimating follicle numbers.