The response surfaces methodology consists of a group of empirical techniques devoted to the evaluation of relationships existing between a cluster of controlled experiment factors and measured responses according to one or more selected criteria (Fereidouni et al., 2009). ECHIP experimental design software (Wilmington, DE) was used to design the experiments and analyze the results of oil extraction stage. To design the experiments, three different factors of particle size, solvent amount and ultrasonication power were selected. The design composed of 19 experimental trials with 5 replications. Five replicate runs were performed to allow the estimation of pure error. All experiments were carried out in a random order to minimize the effect of unexplained variability in the observed responses due to irrelevant factors (Sin et al., 2006). Table 2. shows the independent variables and their levels used in the experimental design. The statistical analyses of direct biodiesel production and in-situ transesterification stages were carried out by measuring the standard deviation for all the results and replicating the trials twice.