The female reproductive system is important as the site for development and fertilization of an oocyte, for implantation and development of an embryo, and for growth and delivery of the fetus. It also produces protein and steroid hormones that help maintain a female's health. Although the female phenotype is the default pathway for the development of the urogenital system, many processes can become disrupted during and after development which may originate from developmental problems. Improper development can be the underlying cause of structural malformations, sub-or infertility, hormonal abnormalities, endometriosis, carcinogenesis, or other detrimental outcomes. Our research programs examine the normal physiology and function of the female reproductive system and how it can become damaged due to pathologies or environmental/ therapeutic exposures, with a focus on the ovary, ovarian follicles, and ovarian hormones. This chapter will describe detailed protocols of an in vitro organ culture system and methods to analyze changes in follicle formation, follicle development, and ovarian physiology. These methods can also be applied to the study of other aspects of female reproduction.