All cells contain proteases, which effect catalytic hydrolysis of the peptide bond between amino acids in the protein backbone. Typically, proteinases are prevented from nonspecific proteolysis by regulation and physical separation into different subcellular compartments; however, this segregation is not retained during cell lysis to release a protein of interest. Prevention of proteolysis during protein purification often takes the form of a two-pronged approach; first the inhibition of proteolysis in situ, followed by the separation of the protease from the protein of interest via chromatographical purification. Proteinase inhibitors are routinely used to limit the effect of the proteinases before they are physically separated from the protein of interest via column chromatography. Here, commonly used approaches to reducing proteolysis during chromatography are reviewed.