The purpose of this protocol is to provide a comprehensive, stepwise guide for assessing mitophagy flux utilizing a live-cell mt-KEIMA approach. The proposed protocol is sensitive, reproducible, quantitative, and easy to perform. While mitophagy has been extensively studied, current methodologies primarily focus on terminal measurements, neglecting the dynamic aspect of this process. Hence, the introduction of this straightforward live-cell mitophagy tracing protocol enables real-time …