Updated project metadata. We present data using a novel method to simultaneously identify and quantify transferred male seminal proteins and the female reproductive proteome using multiplexed Tandem-Mass-Tag (TMT) isobaric labelling of the lower female reproductive tracts dissected from virgin- or recently mated- females of three species of the virilis group. We identified over 200 putative male ejaculate proteins many of which show differential abundance between species. We also identified over 2000 proteins providing the first description of the Drosophila female reproductive tract proteome outside of the melanogaster group which also shows significant divergence between species. We then assessed the utility of species-specific compared to single species query databases for protein identification and quantification.