Traditional drug sensitivity assay confirms the cell death by time-consuming fixation and labeling. This snapshot evaluation overlooks the valuable time-course pharmacodynamics that could offer new insights for accurate drug screening. In this study, we have developed a label-free method to promptly detect cell senescence using endogenous lipofuscin-like autofluorescence. Following drug treatment, we observed that damaged mitochondria release molecules emitting lipofuscin-like red autofluorescence. This corresponding fluorescence intensity significantly increases in apoptotic and necrotic cells. Such innovative approach enables the real-time observation of treatment outcomes in 3D tumor organoids and has the potential to determine drug sensitivity earlier than the Annexin V/PI assay. This metabolic fluorescence signature could substantially enhance the efficiency of drug sensitivity testing.