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Publisher Summary This chapter describes some current protocols used in proteome studies of bacterial pathogens together with some relevant applications. Proteome analysis experiments can be divided into five stages: (1) sample preparation, (2) fractionation, (3) spot detection, (4) spot identification, and (5) protein microsequencing. The proteomes of bacterial pathogens are intimately linked with the use of 2D-GE, which is the only technique capable of resolving the vast majority of expressed proteins, and giving direct information on their patterns of expression. The proteome concept, however, is more general than this and has emerged because of the need to study the ways, in which cellular responses are integrated and to define the roles of the many novel proteins currently being discovered. Just as the initial methods for sequencing genomes are being replaced by ever more rapid procedures that require little human intervention, it is probable that 2D-GE eventually be superseded by methods that combine greater separating power with increased accuracy, speed, and sensitivity of protein detection.