A comparative evaluation of single-stage and two-stage anaerobic digestion processes for biomethane and biohydrogen production using thin stillage was performed to assess the impact of separating the acidogenic and methanogenic stages on anaerobic digestion. Thin stillage, the main by-product from ethanol production, was characterized by high total chemical oxygen demand (TCOD) of 122 g/L and total volatile fatty acids (TVFAs) of 12 g/L. A maximum methane yield of 0.33 L CH4/gCODadded (STP) was achieved in the two-stage process while a single-stage process achieved a maximum yield of only 0.26 L CH4/gCODadded (STP). The separation of acidification stage increased the TVFAs to TCOD ratio from 10% in the raw thin stillage to 54% due to the conversion of carbohydrates into hydrogen and VFAs. Comparison of the two processes based on energy outcome revealed that an increase of 18.5% in the total energy yield was achieved using two-stage anaerobic digestion.