Proteomics is a widely used method for defining the protein composition of a complex sample. As this approach allows for identification and quantification of proteins across a broad dynamic range as well as detection of post-translational modifications, proteomics is an ideal platform to investigate the gut microbiome at a functional level. The gut microbiome is a dynamic environment which is crucial for overall health and fitness. Imbalances in the gut microbiome can influence nutrient absorption, pathogen resistance, inflammation, and various human diseases. Metaproteomic analysis of the gut microbiome is currently being performed on bacteria isolated from (1) fecal samples (2) colonic lavage, or (3) colon biopsies. Investigation of the gut microbiome has demonstrated that within the colon, there are distinct communities based on spatial location, and separable from the gut microbiomes isolated from stool. In addition to expanding our understanding of host-bacterial interactions for human health and disease, gut microbiome analysis is being utilized for biomarker development to discriminate normal individuals and diseased (i.e., inflammatory bowel disease or colon cancer) patients as well as to monitor disease activity and prognosis.