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Most toxic compounds including cancer drugs target mitochondria culminating in its permeabilization. Cancer drug-screening and toxicological testing of compounds require cost-effective and sensitive high-throughput methods to detect mitochondrial damage. Real-time methods for detection of mitochondrial damage are less toxic, allow kinetic measurements with good spatial resolution and are preferred over end-stage assays. Cancer cell lines stably expressing genetically encoded mitochondrial-targeted redox-GFP2 (mt-roGFP) were developed and validated for its suitability as a mitochondrial damage sensor. Diverse imaging platforms and flow-cytometry were utilized for ratiometric analysis of redox changes with known toxic and cancer drugs. Key events of cell death and mitochondrial damage were studied at single-cell level coupled with mt-roGFP. Cells stably expressing mt-roGFP and H2B-mCherry were developed for high-throughput screening (HTS) application. Most cancer drugs while inducing mitochondrial permeabilization trigger mitochondrial-oxidation that can be detected at single-cell level with mt-roGFP. The image-based assay using mt-roGFP outperformed other quantitative methods of apoptosis in ease of screening. Incorporation of H2B-mCherry ensures accurate and complete automated segmentation with excellent Z value. The results substantiate that most cancer drugs and known plant-derived antioxidants trigger cell-death through mitochondrial redox alterations with pronounced ratio change in the mt-roGFP probe. Real-time analysis of mitochondrial oxidation and mitochondrial permeabilization reveal a biphasic ratio change in dying cells, with an initial redox surge before mitochondrial permeabilization followed by a drastic increase in ratio after complete mitochondrial permeabilization. Overall, the results prove that mitochondrial oxidation is a reliable indicator of mitochondrial damage, which can be readily determined in live cells using mt-roGFP employing diverse imaging techniques. The assay described is highly sensitive, easy to adapt to HTS platforms and is a valuable resource for identifying cytotoxic agents that target mitochondria and also for dissecting cell signaling events relevant to redox biology., Highlights • Mitochondrial oxidation is an universal marker for mitochondrial damage and mitochondrial permeabilization. • Ratiometric imaging of mt-roGFP in high-throughput mode allows rapid screening of compounds that target mitochondria. • Real-time ratiometric imaging of mt-roGFP and mitochondrial permeabilization reveals a biphasic redox alteration in cells undergoing mitochondrial permeabilization.