This chapter describes the purification procedure of aerolysin from Aeromonas hydrophila. Aerolysin is a soluble cytolytic protein produced by the gram-negative bacteria Aeromonas hydrophila. It is released to the extracellular medium as an inactive protoxin (proaerolysin) that is converted to the active protein by one or more extracellular proteases which are also exported by A. hydrophila. The mechanism of action of the toxin is very similar to the mechanism proposed for staphylococcal alpha toxin. By far the easiest methods of measuring the activity of aerolysin are based on the ability of the toxin to cause the hemolysis of red cells. Plate assays measure the clearing of red cell suspensions in wells and the absence of cell pellets at the bottom of wells. The presence of trypsin inhibitor at a final concentration of 1 mg/ml in the phosphate buffer solution (PBS)-albumin used for serial dilution has no affect on the results of the assay.