A quantitative assay for measuring surfactin in culture supernatants was developed based on its ability to cause hemolysis. The presence of 145 ml ethanol l(-1) at 37 degreesC in reaction mixture gave an optimal sensitivity. Compared with measurements of dry weights of surfactin, the hemolytic assay gave more reliable concentration values because it did not involve separation steps from the supernatant. The hemolytic assay herein proposed showed to be a simple and low-cost method to readily assess biosurfactants in liquid cultures.