A kinetic estimation method for the evaluation of active biomass concentration of acetate utilizing methanogen in mixed culture is proposed and discussed, based on the Monod model. Batch experiments were conducted using an enrichment culture of acetate utilizing methanogen in three different dilution levels to ascertain availability of the estimation method.“Active” biomass concentrations were kinetically estimated by both acetate consumption and methane production rates. Both estimated biomass concentrations were proportional to the dilution levels of the enrichment culture. The simultaneously estimated maximum specific growth rate was in good agreement with the literature values in the pure cultures. The changes of acetate and methane concentrations with time in the experiments were clearly explained by the Monod model with the estimated values. Then the estimation method was applied to the determination of concentration of acetate utilizing methanogen in a sewage digester sludge. It was shown that the “active”acetate utilizing methanogen occupied only 1-2% of VSS component. From these results, the “active” biomass concentration is shown to be very useful as a biomass index for the evaluation of treatment efficiency in mixed culture systems which contain non-biomass, such as sediments and sludges.