This article describes the use of a simplex centroid mixture experimental design to optimize the fermentation medium in the production of sophorolipids (SLs) using Candida bombicola. In the first stage, 16 media ingredients were screened for the ones that have the most positive influence on the SL production. The sixteen ingredients that were chosen are five different carbohydrates (fructose, glucose, glycerol, lactose, and sucrose), five different nitrogen sources (malt extract, peptone extract, soytone, urea, and yeast extract), two lipid sources (mineral oil and oleic acid), two phosphorus sources (K2HPO4 and KH2PO4), MgSO4, and CaCl2. Multiple regression analysis and centroid effect analysis were carried out to find the sugar, lipid, nitrogen source, phosphorus source, and metals having the most positive influence. Sucrose, malt extract, oleic acid, K2HPO4, and CaCl2 were selected for the second stage of experiments. An augmented simplex centroid design for five ingredients requiring 16 experiments was used for the optimization stage. This produced a quadratic model developed to help understand the interaction amongst the ingredients and find the optimal media concentrations. In addition, the top three results from the optimization experiments were used to obtain constraints that identify an optimal region. The model together with the optimal region constraints predicts the maximum production of SLs when the fermentation media is composed of sucrose, 125 g/L; malt extract, 25 g/L; oleic acid, 166.67 g/L; K2HPO4, 1.5 g/L; and CaCl2, 2.5 g/L. The optimal media was validated experimentally and a yield of 177 g/L was obtained. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 26: 938-944, 2010