Tracking macroautophagic/autophagic flux in live cells is vital for understanding its pathophysiology; however, its dynamic nature complicates assay development. Although fluorescent protein-tagged markers and microenvironment-sensitive small-molecule fluorescent probes have been developed, non-transfection-based and highly specific assays remain underexplored. In this study, we present the design, synthesis, and application of an activity-based autophagy probe (ATP1) for dynamically quantifying autophagic flux. ATP1 was developed through an iterative, docking-guided design strategy to secure MAP1LC3/LC3 engagement, coupled with in-depth analysis of structure-fluorescence relationships to program dual smart-signal behaviors. It displays LC3-binding-triggered fluorogenic activation and autophagosome-lysosome fusion-triggered ratiometric changes. By engaging LC3, the probe is inherently specific to autophagy, and its dynamic signal enables real-time tracking of autophagic flux with high sensitivity. We demonstrate ATP1's exceptional performance in live cells and mice, with a dynamic signal paralleling the mRFP-GFP-LC3 assay. Notably, ATP1 provides significant benefits, including low background signals, compatibility with primary cells, and effectiveness in wild-type mice, where transfection-based assays are often impractical. Furthermore, the probe aids in the discovery of autophagy modulators. In conclusion, ATP1 offers a straightforward, specific, and non-transfection-based method for assessing autophagic flux, serving as a powerful tool for advancing autophagy research.: 3-MA: 3-methyladenine; ATP: autophagy probe; BafA1: bafilomycin A; CBF: cerebral blood flow; FP: fluorescent protein; HBMECs: human brain microvascular endothelial cells; HBSS: Hanks' balanced salt solution; HEPES: 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; ITC: isothermal titration calorimetry; LIR: LC3-interacting region; LSCI: laser speckle contrast imaging; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; OGD: oxygen-glucose deprivation; PBS: phosphate-buffered saline; Rapa: rapamycin; Wort: wortmannin. Abbreviations1
