The removal of chemical substances during wastewater treatment plays a key role in environmental risk assessments. In this study, the usefulness of the activated sludge die-away test (OECD Test Guideline [TG] 314B) was evaluated in terms of its accuracy for predicting the removal of cationic surfactants in wastewater treatment using an ester-amide-type dialkylamine salt (EA). The results show that the estimated removal rate through aerobic biological treatment (37.1%) in activated sludge, calculated from a first-order elimination rate constant, was lower than measured removal rates in actual water treatment systems (wastewater treatment plant [WWTP]: ≥ 99.9%; on-site domestic wastewater treatment plant: ≥ 99.7%). The lower estimation was probably caused by the tendency that chemical substances with high K oc values were transferred from the aqueous fraction to the suspended solid (SS) fraction to activated sludge. Thus, the EA concentration in effluent from the WWTP was calculated from the EA concentration of the SS fraction and the SS concentration in effluent; the estimated removal rate (99.7-99.9%) was equivalent to the measured removal rate. Therefore, when using OECD TG 314B to estimate the removal of chemical substances with a high K oc value, removal via adsorption to activated sludge should be considered.