Publisher Summary This chapter discusses the vacuolar proteases and proteolytic artifacts in Saccharomyces cerevisiae (S.cerevisiae). The yeast S.cerevisiae contains a large number of proteases that are located in various compartments and membranes of the cell. These include endoproteinases, carboxypeptidases, aminopeptidases, and dipeptidylaminopeptidases. Some of the proteases pose significant impediments to the analysis of biochemical processes and/or purification of proteins and can generate artifacts concerning the activity, structure, and intracellular location of proteins. Of the many cellular proteases, lumenal vacuolar proteases probably comprise the major source of problems. Found soluble within the vacuole are endoproteinases A and B, carboxypeptidases Y and S, aminopeptidase I, and aminopeptidase ApY. An additional source of protease problems is Zymolyase because commercial preparations of Zymolyase contain substantial amounts of a protease that may catalyze hydrolysis of azocoll. The chapter describes conditions and procedures that affect the levels and activities of the vacuolar proteases and presents both genetic and biochemical methods for coping with protease problems.