The development of early diagnostics and prevention strategies for preterm birth is an important global health challenge with the potential to impact over 15 million children annually, by improving health outcomes and reducing economic burden. Advances in microbial sequencing technology have opened the door to 16S rRNA gene survey, whole metagenomics, and whole transcriptomics, providing molecular evidence that the composition of the vaginal microbiome affects pregnancy outcomes in women, particularly those of African descent. A current gap in our molecular level understanding of the vaginal microbiome as it relates to healthy pregnancies is the metaproteome which comprises proteins from both the woman and colonizing microorganisms. Herein, I describe the development of a label-free mass spectrometry-based workflow for preparing and analyzing the vaginal metaproteome as sampled from vaginal swab extracts. The workflow was applied to two longitudinal cohorts of vaginal swab samples collected during the VCU MOMS-PI study. The work presented herein demonstrates for the first time that sufficient vaginal-specific biomaterial can be extracted from swabs for metaproteomics analysis as evidenced by high proteome coverage (>1790 human and >1609 microbial proteins), quantitative readouts for over 37% of identified proteins, and the identification of candidate protein biomarkers that change with gestational age and parturition status.