摘要Conventional ex vivo drug screening platforms struggle to recapitulate native subcellular microenvi-ronments,leading to high off-target rates and compromised discovery of bioactive compounds.To address this,we developed subcellular target-tracking fluorescent-visualization-based interaction screening(SubTrack-FVIS),a platform combining super-resolution imaging with target-specific fluo-rescent tagging.SubTrack-FVIS first maps nanoscale spatial distributions of drug targets within living cells,then screens compound libraries to identify molecules specifically binding to target-enriched domains,and finally quantifies drug-target interactions through super-resolution imaging tracking.Compared to traditional toolbox,SubTrack-FVIS reduces off-target effects by evaluating compound binding within native subcellular architectures.When applied to the lysosomal vacuolar H+-ATPases(V-ATPase)subunit,ATP6V1A,a validated anti-cancer target,this approach identified for lysosomal alka-lization fluorescent drug(LAFD)as a potent inhibitor.Super-resolution imaging revealed LAFD's dy-namic binding to ATP6V1A clusters,enabling real-time visualization of V-ATPase inhibition and subsequent lysosomal destabilization.Crucially,SubTrack-FVIS uncovered LAFD's unique mechanism of blocking autophagosome-lysosome fusion,resolving autophagic flux obstruction at sub-100 nm reso-lution.This platform establishes a visualization framework for discovering drugs within physiological subcellular contexts while simultaneously decoding their mechanistic impacts,offering application potential for target-centric drug development.