Abstract. The purpose of this study was to investigate the combined influence of three-level, three-factor variables on the formulation of dacarbazine (a water-soluble drug) loaded cubosomes. Box-Behnken design was used to obtain a second-order polynomial equation with interaction terms to predict response values. In this study, the selected and coded variables X 1 , X 2 , and X 3 representing the amount of monoolein, polymer, and drug as the independent variables, respectively. Fifteen runs of experiments were conducted, and the particle size (Y 1 ) and encapsulation efficiency (Y 2 ) were evaluated as dependent variables. We performed multiple regression to establish a full-model second-order polynomial equation relating independent and dependent variables. A second-order polynomial regression model was constructed for Y 1 and confirmed by performing checkpoint analysis. The optimization process and Pareto charts were obtained automatically, and they predicted the levels of independent coded variables X 1 , X 2 , and X 3 (−1, 0.53485, and −1, respectively) and minimized Y 1 while maximizing Y 2 . These corresponded to a cubosome formulation made from 100 mg of monoolein, 107 mg of polymer, and 2 mg with average diameter of 104.7 nm and an encapsulation efficiency of 6.9%. The Box-Behnken design proved to be a useful tool to optimize the particle size of these drug-loaded cubosomes. For encapsulation efficiency (Y 2 ), further studies are needed to identify appropriate regression model.