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Drug-induced acute kidney injury (AKI), caused by renal drug metabolism, has been regarded as a main problem in clinical pharmacology and practice. However, due to the lack of effective biomarkers and noninvasive real-time tools, the early diagnosis of drug-induced AKI is still a crucial challenge. The superoxide anion (O 2 ˙ - ), the preliminary reactive oxidative species, is closely related to drug-induced AKI. In this paper, we reported two new mitochondria-targeted fluorescent probes for investigating AKI via mapping the fluctuation of O 2 ˙ - with high sensitivity and selectivity by the combination of rational design and a probe-screening approach. Small-molecule fluorescent probes ( Naph-O 2 ˙ - and NIR-O 2 ˙ - ) with high accuracy and excellent selectivity were successfully applied to detect endogenously produced O 2 ˙ - in living cells and tissues by dual-model confocal imaging, and to trap the fluctuation of the O 2 ˙ - level during the drug-induced nephrotoxicity. Moreover, probe NIR-O 2 ˙ - was also used to elucidate the protective effects of l-carnitine (LC) against drug-induced nephrotoxicity for the first time. Therefore, these probes may be potential chemical tools for exploring the roles of O 2 ˙ - in complex nephrotoxicity disease systems.