The bacterial proteome is highly diverse and quickly adapts to physiological stimuli, available nutrients and competition. The gut microbiome is a complex integral part of human physiology and has long been known to play an essential role in digestion and host metabolism. Increasing evidence further highlights its impact on human behavior and it has been associated with a variety of diseases, ranging from inflammatory bowel disease to cancer. Despite the intricate interplay between the microbiome and its host, mechanistic insights into their interactions are scarce and to a large extent based either on metagenomic data or animal studies. Although indispensable for the characterization and analysis of the microbiome on a systems level, these approaches provide only indirect mechanistic information on the subcellular level. Protein annotation in these species is incomplete with information about protein existence, let alone function, being mainly based on predictions. A systematic understanding of protein functions in gut bacteria will not only provide insights into their metabolism, but also contribute to better understanding of the formation, interplay and molecular function of the microbiome in relation to the host. Here, we develop a multiplexed Thermal Proteome Profiling-based approach to map the functional proteome of the gut microbiome species P.vulgatus and B.uniformis. Our approach involves combinatorial treatment of lysates with pools of compounds, allowing us to explore a large number of potential protein-ligand pairs. We have probed the binding partners of 56 compounds, ranging from nutrients to host hormones and drugs and validated our approach using highly conserved processes. Our data provide insights into previously uncharacterized aspects of gut bacterial biology such as sugar sensing, antibiotic transport and response to host hormones.