Within the ovary, anti-Müllerian hormone (AMH) is secreted by the granulosa cells of small growing follicles, and limits the activation of primordial follicles. In a series of studies, it has been shown that supraphysiological levels of AMH can (i) act as a contraceptive, blocking folliculogenesis at the primary stage, and (ii) safeguard the ovary from chemotherapy-induced fertility insults. As such, AMH technologies are attractive tools for the preservation of female fertility. However, the development of AMH technologies is currently limited by both an incomplete understanding of the mechanisms of AMH action, and an inability to manufacture sufficient amounts of AMH for in vivo delivery. Here, we first aimed to improve the production of bioactive AMH. To address this, we used targeted mutagenesis to improve the processing efficiency of pro-AMH. Substitution of the native cleavage site intervening the AMH pro- and mature domains with an ideal processing site increased the yield of mature AMH by up to 5-fold. Next, we aimed to enhance the potency of AMH by improving the ability of AMH to bind to its target receptors. To achieve this, we used targeted mutagenesis to identify loss and gain of function AMH mutants. Using AMH-responsive in vitro assays, we identified a key mutation in AMH that enhanced AMH bioactivity by as much as 5-fold. Excitingly, combining the cleavage site and receptor-binding modifications resulted in an overall 100-fold improvement in AMH bioactivity. These experiments also uncovered key residues in AMH that mediate interactions with the AMH receptors. Ultimately, these studies have improved our understanding of the mechanisms of AMH bioactivity, and enabled the generation of a more potent AMH analogue. Our AMH analogues are attractive tools for preserving female fertility, and also have applications as non-surgical sterility agents in animals.