Dried papaya peels exhibited superior proteolytic activity to the fresh peels. An extraction with phosphate buffer pH 7.0 greatly maintained proteolytic activity when compared to water. SDS-PAGE verified that the extracted dried papaya peels held a wide range of proteins. To optimize the three-phase partitioning (TPP) for isolating the papaya peel proteases required a ratio of crude extract to t-butanol, the (NH4)(2)SO4 concentration and the TPP cycles. The ratio of the crude extract to t-butanol of 1.0:0.5 with the presence of 20% (NH4)(2)SO4 resulted in the highest proteolytic recovery at 253.5%, and 15.8-fold of purification in the bottom phase. The TPP was then optimized by adding up to 55% (NH4)(2)SO4 to the bottom phase of the first step. A purification of 10.1-fold with about 89.4% recovery was obtained. This study showed the TPP can be effectively employed for the extraction of proteases from papaya peels. (C) 2010 Elsevier Ltd. All rights reserved.