This paper reports the results of a study on the analysis and characterization of titanium dioxide (TiO2) nanoparticles (NPs) in samples that were collected from selected freshwater dams in South Africa. The study employed a combination of complementary techniques to ascertain the analytical procedures and the occurrence of TiO2 NPs in water. Characterization, identification and quantification of TiO2 NPs in surface water samples from five sampled dams was performed using several techniques including scanning electron microscopy-energy dispersive X-ray (SEM-EDX), fluorescence excitation–emission matrix (FEEM) and single particle inductively coupled plasma mass spectroscopy (SP-ICP-MS). The SP-ICP-MS technique was able to simultaneously determine the size and concentration of both the dissolved and particulate titanium (Ti) in water samples. The Ti-containing NPs were found at concentrations ranging from 8.3 × 104 parts/mL to 1.4 × 105 parts/mL in the presence of 14.9 μg/L to 243 μg/L dissolved Ti. The TiO2 nanoparticles were mostly composed of small agglomerates ranging from 102.9 nm to 158.9 nm in size.