ABSTRACT Characterising bioflocculation and settling in activated sludge processes is mandatory to better understand and optimise organic matter removal and capture. Among the few indicators available, Threshold Of Flocculation (TOF) reflects the ability of activated sludge to settle following Stokesian settling mechanisms. In this work, the original method to determine TOF has been improved to gain accuracy and reproducibility. An R-script was proposed for systematic analysis of the experimental curves representing the total suspended solids supernatant concentration as a function of the initial concentration of the total suspended solids. The strength of this new method is to base curve analysis on the settling mechanisms that occur in the experimental setup. Two thresholds are mathematically identified, corresponding to (i) the solid concentration required to induce discrete flocculent settling and (ii) the minimum concentration to form large flocs that interact with each other (TOF). The novel method was successfully applied to different types of sludge (HRAS, CAS, and densified sludge).