Autophagy is a natural mechanism in which the cell removes and degrades cellular components with autolysosomes. It is a popular research area because autophagy is related to many physical and pathological processes. The 2016 Nobel Prize in Physiology is granted to Yoshinori Ohsumi for his contribution in autophagy. In autophagy studies, LC3-I and LC3-II detection is a must-have experiment to track the autophagy process. Therefore, we would like to share five important notes for quantifying autophagy with LC3.