In situ transesterification of biomass has the potential to simplify and reduce the cost of biodiesel production from microalgae. This route has the advantage of reducing the number of unit operations, by avoiding the lipid extraction step, prior to transesterification reaction. Thus, the biomass is directly contacted with the alcohol and the catalyst required to convert lipids into biodiesel. In this study, essential process parameters for acid in situ transesterification of microalgae biomass to FAME (fatty acid methyl esters) production were evaluated. Parameters such as the particle size of dry microalgae conglomerates and cosolvent use in the reaction mixture were studied, besides of the temperature and catalyst dosage. An experimental BoxBehnken design was used for the optimization of the FAME yield. The best FAME yield obtained under optimized conditions was of 80% wt, using a microalgae particle size fraction of <150 mu m, a catalyst dosage of 125% wt, and a reaction temperature of 67 degrees C and adding 27% vol/vol of petroleum ether as cosolvent in the reaction mixture. According to the obtained results from experimental design, the FAME yield rose with both when the particle size decreased and the cosolvent proportion in the reaction mixture increased. Moreover, adding a cosolvent increased the saturated and monounsaturated methyl ester content in the sample, improving its stability.