Over the last two decades, sequencing-based methods have revolutionised our understanding of niche-specific microbial complexity. In the lower female reproductive tract, these approaches have enabled identification of bacterial compositional structures associated with health and disease. Application of metagenomics and metatranscriptomics strategies have provided insight into the putative function of these communities but it is increasingly clear that direct measures of microbial and host cell function are required to understand the contribution of microbe–host interactions to pathophysiology. Here we explore and discuss current methods and approaches, many of which rely upon mass-spectrometry, being used to capture functional insight into the vaginal mucosal interface. In addition to improving mechanistic understanding, these methods offer innovative solutions for the development of diagnostic and therapeutic strategies designed to improve women’s health.