Autophagy is a key process that maintains cellular homeostasis. Autophagy contributes to various physiological and pathophysiological processes. Development of methodologies for autophagy detection has greatly facilitated the research on autophagy. Among these methodologies, GFP-LC3 reporter has been popularly used in the literature. In this chapter, we will detail step-by-step the GFP-LC3 reporter protocol we have adapted in our lab. This protocol begins with the generation of lentivirus expressing GFP-LC3. Then, the cells are transduced with titrated virus. After selecting the positive cells, single colonies are isolated, characterized, validated, and used in further study.