Mitochondrial production of O₂^•– and H₂O₂ has been implicated in redox signaling and in the pathogenesis of numerous diseases including cancer, neurodegeneration, and cardiovascular diseases. To understand the exact role of those species, new chemical biology tools for selective and efficient induction of mitochondrial superoxide production are needed. Here, we report the development of a new viologen-based redox cycling agent, mito-diquat (Mito-DQ), capable of inducing targeted mitochondrial O₂^•– production at significantly higher rates as compared to previously reported mito-paraquat (Mito-PQ), a widely used chemical tool to study mitochondria-dependent redox signaling.