A procedure has been developed for the determination of non-ionic surfactants (NS) adsorbed on particles of alive and dead activated sludge. The procedure also enables the determination of adsorption of major biodegradation by-products: short-chained ethoxylates, long- and short-chained PEG. The basis of measurement is the determination of NS concentration in a slurry of activated sludge and in a solution phase. The difference between these two concentrations represents the NS adsorbed on activated sludge. Separation of NS and their biotransformation by-products from samples and then on narrower fractions was performed by a sequential liquid-liquid extraction and precipitation with modified Dragendorff reagent. The indirect tensammetric technique (ITT) was applied for the final determination. The developed method was checked using the example of the treatment of the surfactant C12E10 (oxyethylated fatty alcohol) (C12E10) in the continuous flow activated sludge facility. No statistically significant accumulation of C12E10 on the alive activated sludge was detected, probably because of faster C12E10 fission than its adsorption. However, significant adsorption of the short-chained ethoxylates (including free alcohol) on the alive activated sludge was found, as well as statistically significant adsorption of long- and short-chained PEG. The adsorption of surfactant C12E10 and its biodegradation by-products on dead activated sludge was found to be higher than the species adsorption on alive activated sludge.