Dysfunctional mitochondria accumulate in many human diseases. Accordingly, mitophagy, which removes these mitochondria through lysosomal degradation, is attracting broad attention. Due to uncertainties in the operational principles of conventional mitophagy probes, however, the specificity and quantitativeness of their readouts are disputable. Thorough investigation of the behaviors and fates of fluorescent proteins inside and outside lysosomes enabled us to develop an indicator for mitophagy, mito-SRAI. Through strict control of its mitochondrial targeting, we were able to monitor mitophagy in fixed biological samples more reproducibly than before. Large-scale image-based high-throughput screening led to the discovery of a hit compound that induces selective mitophagy of damaged mitochondria. In a mouse model of Parkinsons disease, we found that dopaminergic neurons selectively failed to execute mitophagy that promoted their survival within lesions. These results show that mito-SRAI is an essential tool for quantitative studies of mitochondrial quality control. Display omitted •mito-SRAI provides reliable mitophagy readouts under both live and fixed conditions•mito-SRAI uses a fluorescent protein that is resistant to lysosomal environments•High-throughput screening led to the discovery of mitophagy-inducing compounds•Evidence was given for loss of nigral dopaminergic neurons due to mitophagy failure Through investigation of the behaviors and fates of fluorescent proteins inside and outside of lysosomes, Katayama et al. developed a signal-retaining autophagy indicator, which they localize to mitochondria (mito-SRAI) to quantitatively measure mitophagy. They apply their new tool in a high-throughput in vitro screen for chemical inducers of mitophagy and in a mouse model of Parkinsons disease.