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 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). In combination with an update and expansion of the seminal vesicle sperm proteome and sex-specific isotopic labeling to identify female-contributed sperm proteins, our data provide a comprehensive, quantitative analysis of the molecular life history of Drosophila sperm.