The biological conversion of biomass in Anaerobic Digestion (AD) into methane was studied by many researchers in recent years. In the present study, optimization of methane production during chemical oxygen demand (COD) removal was observed in a novel Multi-Stage Anaerobic Reactor (MS-AR). A synthetic glucose was used as a feed substrate and the reactor was operated at a hydraulic retention time (HRT) of 1 to 4 d. Two complementary test procedures for methane optimization were evaluated; the theoretical and experimental. The theoretical methane gas was recorded as 50.13, 50.02, 50.16, and 50.22 % for HRT of 4, 3, 2 and 1 day, respectively. The results signify well with the empirical formula at each HRTs studied in the reactor. However, the quantity of methane gas present in the real application is significantly lower than the theoretical. This is due to the microorganism activity in the reactor that may have interfere with the efficiency of the biogas production. Actual data showed a decrease in the methane gas production (35.4, 21.2, 19.8, and 18.4 %) in the reactor. Thus, theoretical formula together with the actual data provides alternative method for the evaluation of bioenergy potential in AD
