The objective of this work is to estimate active nitrifying biomass and its main influencing factors in low-loaded biofilters based on operational data. An analytical approach based on balancing growth, decay and biomass removed by backwashing is proposed. The method is developed and applied in pilot-scale rapid sand filters for drinking water treatment. Decay rate was measured directly in the filter for different temperatures. To assess the amount of active biomass in backwash water, a technique based on respiration measurements was used. Backwash losses increased overproportional with balanced biomass in the filter. The impact of both parameters on active biomass is quantified exemplarily for a given constant nitrification rate.