All living organisms utilise reactive oxygen species (ROS) for essential biological functions, many of which involve signal-transduction pathways, including for regulation of the activity of enzymes, signalling cell growth and differentiation, and mediation of inflammation. Aberrant characteristics of the tumour microenvironment, which include acidosis, hypoxia, inflammation, and vascular abnormalities, can result in differences in ROS between cancer cells and healthy tissues. Altering intracellular ROS, including the superoxide anion (•O2–), hydroxyl (•OH), singlet oxygen (1O2), and hydrogen peroxide (H2O2), has been proposed to induce cancer cell apoptosis resulting from ROS-damage of biomolecules, including lipids, proteins, and DNA. Intracellular ROS levels in cancer cells can be modulated by nanoparticles in two distinct ways to induce anti-cancer effects. In the first case, the antioxidant properties of nanoparticles are used to lower ROS levels in tumour cells, leading to cell-cycle arrest followed by apoptosis. In the second case, prooxidant nanoparticles are used to elevate ROS levels above tolerable thresholds, leading to increased apoptosis of cancer cells. While higher intracellular ROS levels in cancer cells, which are generated by mitochondrial dysfunction, can promote tumorigenesis, metastasis, and angiogenesis, the excessive accumulation of ROS induces cell death. Therefore, the imposition of specific ROS levels in tumour cells through agents, such as nanoparticles, is a potential strategy for cancer therapy. Detecting ROS alterations in living biological systems is challenging owing to the lack of specificity of ROS-sensitive probes, the high turnover of ROS species, and insufficient sensitivity of detection techniques. 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) is a commonly used ROS probe which, upon cellular uptake, is cleaved into the highly fluorescent 2',7'-dichlorofluorescein (DCF) molecule. In this sense, it represents an indicative H2O2-detection probe for intact cells. However, the range of ROS that can be detected by this probe is not limited solely to H2O2 and so has much greater capabilities. Most of the protocols for ROS measurement using confocal microscopy imaging focus exclusively on the detection of ROS but do not provide quantitative data. The current work presents development of this fluorescent molecule whose fluorescence signal increases in presence of exogenous agents such as the nanoparticles used for the therapeutic applications. We employ advanced confocal fluorescence microscopy and image analysis to quantify ROS signal in cells, demonstrating the efficacy of nanoparticle treatment.