The superoxide radical (O2∙−) is the primary oxygen radical, predominantly formed during the process of oxidative phosphorylation in mitochondria, and it initiates a cascade of other reactive oxygen species (ROS) production in the cell. An imbalance between ROS production and the biological system's ability to detoxify them and repair the resulting damage leads to oxidative stress (OS), which is associated with the development of many diseases. Due to the assumption that O2∙− could be potentially useful in assessing OS levels and the fact that measuring O2∙− could have diagnostic purposes, the aim of this study was to optimize a method for measuring O2∙− in cells using a fluorescent microscope. The fluorescent probe dihydroethidium (DHE) was used for detecting O2∙−, and as experimental model, buccal cells were used. In the first step of the research, the method for measuring O2∙− in cells was optimized in terms of DHE concentration and cell incubation time with DHE. Results indicate that the optimal conditions for measuring O2∙− in cells are 10 minutes incubation of cells at room temperature (25ºC) with a DHE solution at a concentration of 20 μM for the most optimal cytoplasmic staining. Subsequently, the optimized method was applied to measure O2∙− in cells treated with H2O2 to induce OS. It was confirmed that H2O2 is a successful inducer of OS, meaning that it effectively enhances the production of O2∙− in cells. Elevated levels of O2∙− in buccal cells were induced after 10 minutes incubation of the cells with a 30 mM H2O2 solution. The developed method can be applied to monitor O2∙− levels and, consequently, OS in cells.