Titanium dioxide nanoparticles (TiO2 NPs) are one of the most widely used nanomaterials that have been manufactured worldwide and applied in different commercial realms. The well-recognized ability of TiO2 to promote the formation of reactive oxygen species (ROS) has been extensively studied as one of the important mechanisms underlying TiO2 NPs toxicity. As the “gold standard” method to quantify and identify ROS, electron spin resonance (ESR) spectroscopy has been employed in many studies aimed at evaluating TiO2 NPs safety. This review aims to provide a thorough discussion of current studies using ESR as the primary method to unravel the mechanism of TiO2 NPs toxicity. ESR spin label oximetry and immune-spin trapping techniques are also briefly introduced, because the combination of spin trapping/labeling techniques offers a promising tool for studying the oxidative damage caused by TiO2 NPs.