The plethora of data now available from bacterial genome sequencing has opened a wealth of new research opportunities. Many of these have been reviewed in preceding chapters. Genomics alone, however, cannot capture a biological snapshot from an organism at a given point in time. The genome itself is static, and it is the changes in expression of genes, leading to the production of functional proteins, which allows an organism to survive and adapt to a constantly changing environment. Proteomics is the term used to describe the global analysis of proteins involved in a particular biological process. Such processes may be analyzed via comparative studies that examine bacterial strain differences, both phenotypic and genetic, bacteria grown under nutrient limiting conditions, growth phase, temperature, or in the presence of chemical compounds, such as antibiotics. Proteomics also provides the researcher with a tool to begin characterizing the functions of the vast proportion of “hypothetical” or “unknown” proteins elucidated from genome sequencing and database comparisons. For example, study of protein-protein, protein-ligand, protein-substrate, and protein-nucleic acid interactions for a given target protein may all help to define the functions of previously unknown proteins. Furthermore, genetic manipulation combined with proteomics technologies can provide an understanding of how gene expression is regulated. This chapter examines the technologies used in proteome analysis and the applications of proteomics to microbiological research, with an emphasis on clinically-relevant bacteria.