Autophagy is a cellular pathway that regulates the degradation and recycling of proteins and organelles. This mechanism is initiated with the formation of a phagophore, a cup-shaped double membrane that engulfs the cytoplasmic material to be degraded. The phagophore is then elongated and sealed to generate an autophagosome that will be fused with a lysosome, thereby delivering the cargo for degradation. Several ubiquitin-like proteins have been shown to have a crucial role in regulating autophagosome generation and its fusion with the lysosome. One of these proteins is LC3-I, is a soluble cytosolic protein that associates with the autophagosome after C-terminal conjugation to a phosphatidylethanolamine unit (LC3-II). This membrane association is reversibly modulated by the cysteine protease atg4, which deconjugates PE from LC3-II, thus recycling the soluble LC3-I. Detailed studies of the molecular mechanism regulating autophagosome formation and the role of LC3-I and LC3-II are unknown, mainly due to the lack of tools enabling a profound characterization of these processes. Herein, we propose the generation of semisynthetic lipidated LC3 proteins using a chemical biology approach. The fully modified proteins should be unvaluable tools that will contribute to the characterization of the factors regulating autophagy.