Abstract When making biodiesel, slow separation of glycerol; the main by-product of the transesterification reaction, could lead to longer operating times, bigger equipment and larger amount of steel and consequently increased production cost. Therefore, acceleration of glycerol/biodiesel decantation could play a significant role in economic viability of the overall biodiesel refinery process. In the present study, the interactive effects of prominent parameters i.e. temperature (25–65°C), NaCl addition (0–2g/100ml), and methanol concentrations (10–30vol.%) on decantation behavior of glycerol/biodiesel mixture were studied using Box–Behnken design matrix and response surface methodology (RSM). The findings revealed that in low temperature ranges, the main factor influencing decantation speed was density difference while at high temperature ranges, viscosity variation played the major role. Moreover, decantation time was significantly decreased by 200% (3min) at the optimum conditions achieved i.e. 45°C, 1g NaCl addition and 20% excess methanol. The methyl ester yield was measured at >90% under optimum conditions which was increased to ASTM quality (>96.5%) after washing and drying procedures.