This study identified the effect and importance of the start-up period on methane production. Methane production was studied during the start-up period of semi-continuous stirred tank reactor (SCSTR) of anaerobic suspended growth under mesophilic conditions (37±3°C) with semi-continuous feeding. Two reactors were seeded with an inoculum of rumen fluid (5% and 10%) and mixed with anaerobic granular sludge (95% and 90%) as a substrate, respectively. The anaerobic digesters were filled up with 800 ml of these seeds during feeding phase. A supernatant of raw Palm oil mill effluent (POME) was fed in semi-continuous mode during the stabilization phase. The pH of the digesters was kept within the range of 7.2 -7.5 without any adjustment during this period. The effects of different concentration of seeding, MLVSS and COD (mg/L) on the quantity of produced methane during the start–up period were studied. The results showed that during the feeding phase methane content was 82.10% and 81.34% for both mixtures. But at the end of start-up period both reactors have the same percentage which is around 55%. Higher methane production occurred during the feeding phase for the second mixture. MLVSS and COD concentrations have a direct relationship with the methane obtained.