Cellulose nanocrystals (CNCs) are bio-based building blocks for sustainable advanced materials with prospective applications in polymer composites, emulsions, electronics, sensors, and biomedical devices. However, their high surface area-to-volume ratio promotes agglomeration, which restrains their performance in size-driven applications, thereby hindering commercial CNC utilization. In this regard, ultrasonication is commonly applied to disperse CNCs in colloidal suspensions; however, ultrasonication methodology is not yet standardized and knowledge of the effects of ultrasound treatments on CNC size distribution is scarce. This limits the ability to tailor CNC performance in advanced materials. Herein, colloidal suspensions of sulfated CNCs were treated with different ultrasound energy densities up to 40 kJ g−1 CNC and then size-fractionated with asymmetrical flow field-flow fractionation (AF4). On-line multi-angle light scattering and ultraviolet spectroscopy along with off-line dynamic light scattering were used to determine the effect of ultrasonication on particle size distribution. High energy densities facilitated cumulative dispersion of CNC clusters and mean particle length decreased logarithmically with increasing energy density. The suspension’s electrical conductivity concurrently increased, which has been attributed to faster diffusion of smaller particles and exposure of previously obscured surface charges. Colloidal stability, investigated through electrical AF4 (EAF4) and electrophoretic light scattering, was not affected by ultrasonication. Occurrence of minor CNC agglomeration at low ultrasound energy densities over the course of six months suggest the effect was not unmitigatedly permanent.