Currently, protocols for the dispersal of titanium dioxide (TiO2) nanoparticles are not standardized and often yield non-uniform particles and/or insufficient dispersal in liquid medium. Our study aimed to improve dispersal so that TiO2 nanoparticles are of uniform size, making nanotoxicity testing more reliable. Various combinations of vehicles, sonication durations, and sonication volumes were assessed for optimizing preparations of TiO2 nanoparticles. We tested each of five vehicles: ultrapure water (UPW), 0.2% disodium hydrogen phosphate (DSP), Dulbecco's phosphate-buffered saline (PBS), 0.9% saline (S), or S containing 0.05% Tween 80 (ST). We also assessed two sonication durations and three sonication volumes. Each suspension underwent ultrasonication and centrifugation; the supernatants were then analyzed. Particle size was measured by dynamic light scattering. P25 nanoparticles (~100 nm; the type of TiO2 nanoparticles used in our study) in UPW and 0.2% DSP were effectively dispersed; however, those in PBS, S, or ST were not. Relevant duration time and volume for sonication were examined with 0.2% DSP. A sonication time of 30 min and volume of 10 mL for each vial were determined to be optimal sonication conditions as determined with our dispersal assay. Under these optimal conditions, P25 nanoparticles sonicated/centrifuged in UPW or 0.2% DSP remained dispersed and exhibited long-term stability (90 days). We thus have developed a reliable procedure for preparing TiO2 nanoparticles in liquid-phase dispersions for toxicity testing.