The Mississippi gopher frog (MGF, Lithobates sevosa) is a critically endangered amphibian which requires the use of assisted reproductive technologies (ART) to successfully reproduce in captivity. Several challenges to successfully breeding MGFs were investigated to improve reproduction in this species. Accurate identification of biological sex is a necessary first step to reproducing species, thus several non-invasive techniques for sex identification were compared in the weakly dimorphic MGF. Reliably obtaining eggs from female MGFs has also proved difficult, therefore, four exogenous hormone therapies were compared for their success to induce ovulation. Ultrasound imaging was used alongside hormone treatments to improve understanding of ovarian response to hormone treatment. Lastly, short-term cold storage of spermic urine was studied to determine the feasibility of this technique for MGF gamete storage. Findings from these research projects are important for the successful conservation of the MGF by aiding in the production offspring for future reintroduction programs.