•Optimum ultrasound duration for TiO2–water nanofluid is 150min.•Initial particle size is more effective than sonication time on stability.•Comparison with literature and statistical analysis provide useful conclusions. Nanofluids are promising in many fields, including engineering and medicine. Stability deterioration may be a critical constraint for potential applications of nanofluids. Proper ultrasonication can improve the stability, and possibility of the safe use of nanofluids in different applications. In this study, stability properties of TiO2–H2O nanofluid for varying ultrasonication durations were tested. The nanofluids were prepared through two-step method; and electron microscopies, with particle size distribution and zeta potential analyses were conducted for the evaluation of their stability. Results showed the positive impact of ultrasonication on nanofluid dispersion properties up to some extent. Ultrasonication longer than 150min resulted in re-agglomeration of nanoparticles. Therefore, ultrasonication for 150min was the optimum period yielding highest stability. A regression analysis was also done in order to relate the average cluster size and ultrasonication time to zeta potential. It can be concluded that performing analytical imaging and colloidal property evaluation during and after the sample preparation leads to reliable insights.