Two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry (MS) is the classic proteomics approach used to monitor the dynamics of protein abundance and posttranslational modifications in biological systems. In this chapter, we provide detailed protocols for 2-DE-based proteomics of mycobacteria. Adequate standard operating procedures for mycobacterial culture, subcellular fractionation, and selective enrichment of proteins are indispensable prerequisites for targeted proteome analyses. Therefore, we also provide approved protocols for selective and efficient extraction of cytosolic, secreted, and hydrophobic plasma membrane proteins of mycobacteria, as well as for isolation of mycobacteria from infected macrophages.