Compared to other typical cleaning agents, application of enzyme in cleaning of membranes fouled with protein solution promised the high cleaning efficiencies with lower environmental impact. This paper is focused on the mechanisms of protein removal by enzyme cleaning agent from the membrane surface by analysis hydraulic resistance, total protein removal using Lowry method, and membrane surface analysis using MALDI-MS and gel electrophoresis to estimate the foulant composition. Using single and binary protein solutions of bovine serum albumin and beta-lactoglobulin as the feed solution for filtration process, the experimental results indicate that optimum cleaning time and cleaning agent concentration is due to the competition between foulant removal and deposition of enzymes on the membrane during the cleaning process. The removal rate of different protein species in the fouling layer is varied, indicating that cleaning strategies can be tailor-made for fouling layer with different protein compositions. (C) 2008 Elsevier B.V. All rights reserved.