Mitochondrial reactive oxygen species (mtROS or mROS) are generated within mitochondria, primarily during oxidative phosphorylation at the electron transport chain (ETC) on the inner mitochondrial membrane. Electron leakage from complexes I and III partially reduces oxygen to superoxide, which is dismutated to hydrogen peroxide by SOD2 in the mitochondrial matrix and SOD1 in the intermembrane space. Low levels of mtROS are essential for metabolic adaptation and inflammatory response regulation, while elevated mtROS can induce apoptosis, autophagy, cellular senescence, and aging. Recent findings implicate mtROS production in lung monocytes and macrophages in COVID-19 pathogenesis, highlighting mtROS as a potential therapeutic target for novel coronavirus treatments. MitoROS Brite™ NIR 780 is a hydrogen peroxide-sensitive fluorescent probe specifically designed for targeting mitochondria in live cells. Upon oxidation by hydrogen peroxide anions, it emits near-infrared fluorescence, allowing precise quantification of mitochondrial hydrogen peroxide levels via fluorescence microscopy or flow cytometry. Its high cell permeability and mitochondrial selectivity ensure specific detection of hydrogen peroxide with minimal cross-reactivity to other ROS or reactive nitrogen species (RNS). The retention of the oxidized fluorophore within cells makes MitoROS Brite™ NIR 780 invaluable for investigating oxidative stress-related pathologies. Furthermore, its compatibility with multiplexing techniques facilitates concurrent use with other fluorescent mitochondrial probes, enabling comprehensive analysis of multiple mitochondrial parameters within the same cell population. For instance, it can be combined with MitoLite™ Green FM to assess ROS levels and mitochondrial mass or morphology, or with TMRE or TMRM to measure both mitochondrial membrane potential and ROS levels, advancing research into mitochondrial-associated diseases.