Interactions between sperm and the female reproductive tract (FRT) are critical to fertility, but knowledge of the molecular mechanisms by which the FRT interacts with sperm and influences sperm fate remains limited. Here, we used sex-specific isotopic labeling to identify female-derived proteins that contribute to the sperm proteome after long-term storage in the female’s sperm storage organs. In combination with whole-cell quantitative proteomics to track the protein composition of sperm across three female reproductive tissues (bursa, seminal receptacle, and spermatheca) and three post-mating timepoints (30 minutes, 2 hours, and 4 days), as well as an expansion of the seminal vesicle sperm proteome, our data provide a comprehensive, quantitative analysis of the molecular life history of Drosophila sperm.