Cytotoxic T lymphocyte (CTL)-mediated immune responses rely on the efficiency of MHC class I ligand generation and presentation by antigen presenting cells (APCs). Whereas the abnormal expression of MHC molecules and transporters associated with antigen processing (TAPs) are commonly discussed as factors that modulate antigen presentation, much less is known about possible regulatory mechanisms at the level of proteolysis responsible for the generation of antigenic peptides. The ubiquitin-proteasome system is recognized as the major component responsible for this process in the cytosol and its activity can be regulated by cytokines, such as IFN-gamma. However, new evidence suggests the involvement of other proteases that can contribute to cytosolic proteolysis and therefore, to the quality and quantity of antigen production. Here, we review recent findings on an increasing number of proteolytic enzymes linked to antigen presentation, and we discuss how regulation of cytosolic protease activities might have implications for immune escape mechanisms that could be used by tumor cells and pathogens.