It is important to determine the assembly configuration of engineered nanomaterials (ENMs) because assembly configuration influences their fate and transport behavior in the aquatic environment. Aggregated particles are more subject to segregation upon changes of environmental conditions (and vice versa) than agglomerated particles. As a strategic tool for investigating the time-resolved reversible segregating and assembling behavior of ENMs and thus estimating their assembly configuration, a controlled sonication process was proposed. It was hypothesized that the unique colloidal response of ENMs to sonication, with respect to changes in size, might be their intrinsic property associated with assembly configuration. As a model ENM, three different TiO2 particles with unique properties (commercial P-25 and UV 100 and home-made (HM) TiO2) were examined with programmed sonication processes under various environmental settings. When they were dispersed in water, all TiO2 particles tested obviously assembled to form much larger clusters. Size of P-25 decreased immediately upon sonication and did not change under the subsequent quiescence step while sizes of UV 100 and HM gradually decreased and then slowly recovered back to their initial sizes. The trend was generally observed in all conditions tested. The unique colloidal response of TiO2 could be explained by its properties associated with assembly configuration.