The proteomic approaches have considerably evolved over the past two decades. This opened the doors for larger scale and deeper explorations of cellular physiology. Like for other living organisms, using the tools of proteomics has undoubtedly improved knowledge about the foodborne pathogen Listeria monocytogenes. Among the different technologies and approaches permanently evolving in the field of proteomics, the 2-DE is an analytical separation method of choice to resolve thousands of proteins simultaneously in a single gel, allowing their quantification, the study of their posttranslational modifications and the understanding of their biological function. In this, 2-DE remains a perfectly complementary technique to the new high-throughput techniques such as shotgun proteomics approaches. Moreover, in order to gain in analysis depth and improve knowledge about the target of action and the function of proteins in relation to their subcellular location, it is necessary to explore more specifically the different subcellular proteomes. Thus, the subproteomic analyses became essential and dramatically increased these last years, particularly on proteins secreted into the extracellular milieu, named exoproteome, or on cell envelope proteins (cell wall and membrane proteins) which are involved in the interactions with the surrounding environment. Here, the extraction and separation of L. monocytogenes subproteomes are described based on cell fractionation and 2-DE techniques. This chapter gives a workflow to obtain the exoproteome, the intracellular proteome, the cell wall, and membrane proteomes of the Gram-positive bacterium L. monocytogenes. The different steps of 2-DE technology, composed of a first dimension based on the separation of proteins according to their charge, an equilibration step, then a second dimension based on the separation of proteins according to their mass, and finally the staining of proteins in the gel are detailed. Emerging technologies to extract the exoproteome or the cell surface proteome after enzymatic shaving and to analyze them by shotgun method are also discussed briefly.