Autophagy is a self-degradative process required for cell survival and cellular metabolic homeostasis in response to various stresses such as nutrient deprivation, damaged organelles and uncontrolled production of reactive oxygen species (ROS). Autophagy can be activated as a cytoprotective mechanism but excess autophagy is related with cell death. Various medicines including nonsteroidal anti-inflammatory drugs (NSAIDs) are thought to be related with harmful effects on autophagy process. Elucidation of how each drug controls autophagy and how the change of autophagy process induces cell death is important for drug safety evaluation. In this study, we try to set up a strategy to analyze quantitatively the effect of each medicine on autophagic flux and cell death with the use of cellular imaging. In general, autophagic flux consists of three steps: initiation, maturation and completion─fusion of autophagosomes with lysosome and their degradation. Inhibition or overactivation of one of these steps disrupts cellular homeostasis and metabolic system, inducing cell death. Here, we show the comprehensive study for the quantifiable measurement of autophagic flux in a human liver cell line. To quantify autohphgic flux, we analyzed autophagosome spots and autolysosome spots in HepG2 cells infected with mCherry-GFP-LC3 adenovirus. Moreover, spot size (diameter and parameter) analysis was performed to monitor degradation process. We also measured an initiation step of autophagy─formation using analysis of phosphatidylinositol 3-phosphate positive spots with mCherry-WDFY reporter. To analyze the cytotoxicity of drugs, we imaged and quantified the dynamic structural change of key cellular organelles such as the Golgi and mitochondria. Here, we propose an analytical method to differentiate the effects of Rapamycin (an autophagy inducer), Bafilomycin A1 (a V-ATPase inhibitor), SAR405 (a VPS34 inhibitor) and Diclofenac (a NSAID) on autophagic flux. This study can apply to investigating the mode of action of unknown drugs on autophagy and their cytotoxicity.