In insects, female post-mating responses such as increased ovulation are initiated by male seminal fluid proteins (Sfps) and then executed by female pathways and molecules. In Drosophila melanogaster, the Sfp ovulin promotes ovulation by activating female octopamine (OA) neuronal signaling shortly after mating. While ovulin’s effects are well characterized, the broader female molecular machinery governing ovulation remains incompletely understood. Exploring these female mechanisms is important for understanding reproductive physiology and gaining insight into the evolution of reproductive processes. Several reproductive proteins evolve rapidly due to sexual selection, sexual conflict, or relaxed constraint. Ovulin exemplifies this pattern, being among the most rapidly evolving proteins in D. melanogaster. Yet, despite OA signaling’s significant role, the evolutionary patterns of OA receptors remain largely unexplored. Therefore, this dissertation investigates three interconnected questions: (1) What female membrane proteins regulate post-mating ovulation? (2) What female receptor(s) mediate ovulin’s effects? (3) How have OA receptors evolved across Drosophila species? Using ovulin as a probe, we performed evolutionary rate co-variation and AlphaFold-Multimer screens to identify candidate female membrane proteins that are in the ovulation pathway and/or associated with ovulin. Ovulation assays comparing knockdown/knockout females to controls revealed seven regulators of ovulation, Lgr3, GabaβR1, SIFaR, mthl9, Smog, Cirl, and CG6067, acting via ovulin-dependent or -independent pathways. Neuron-specific knockdowns and Gal4-driven expression analyses indicated that several of them function in OA neurons. Notably, Smog shows evidence of positive selection in ligand-binding domains within the melanogaster group, suggesting possible interaction with a rapidly evolving ligand like ovulin. This prompted further screening for ovulin receptor(s) using yeast two-hybrid and in vitro TANGO assays, which, along with prior genetic interaction data, identifying five top candidates: Lgr3, GabaβR1, Smog, Octβ2R, and ETHR. Regarding OA receptor evolution, most are found as single-copy genes across species. While four receptors appear conserved or neutrally evolving, Octβ2R and Octβ3R exhibit recurrent positive selection in the melanogaster group, though in distinct functional regions. Interestingly, Octβ2R does not show evidence of positive selection in the virilis-repleta group and in mosquitoes, suggesting lineage-specific selection pressures. Together, these findings provide a better understanding of the female molecular mechanisms of ovulation and reveal the evolutionary dynamics of key receptors in insect reproduction.