Mitophagy uses lysosomal degradation to remove defective mitochondria. Although probes to detect and measure mitophagy such as mt-mKeima have been developed, they can only image live specimens due to their reliance on a pH difference inside and outside the lysosome. To develop an improved mitophagy probe, Katayama et al. screened a library of fluorescent proteins and identified a cyan-emitting protein called TOLLES that is resistant to lysosomal environments. They used TOLLES as a FRET donor and a YFP variant (YPet) as a FRET acceptor to develop a ratiometric autophagy indicator called SRAI. Starvation of cells to induce autophagy resulted in movement of SRAI to the lysosome, where YPet is degraded, producing a high TOLLES/YPet ratio that was also retained in fixed samples. A mitochondrially targeted variant, mito-SRAI, could detect mitophagy induced by the uncoupler FCCP and Parkin expression and enabled the identification of a compound that enhances mitophagy on damaged mitochondria while sparing intact mitochondria. Overall, the development of mito-SRAI as a reliable readout of mitophagy offers the potential for further understanding of how defects in mitophagy can result in diseases.