AbstractA growing body of evidence suggests that autophagy inhibition enhances the effectiveness of chemotherapy, especially in difficult‐to‐treat cancers. Existing autophagy inhibitors are primarily lysosomotropic agents. More specific autophagy inhibitors are highly sought‐after. The microtubule‐associated protein 1A/1B light chain 3B protein, LC3B, is an adapter protein that mediates key protein‐protein interactions at several points in autophagy pathways. In this work, we used a known peptide ligand as a starting point to develop improved LC3B inhibitors. We obtained structure‐activity relationships that quantify the binding contributions of peptide termini, individual charged residues, and hydrophobic interactions. Based on these data, we used artificial amino acids and diversity‐oriented stapling to improve affinity and resistance to biological degradation, while maintaining or improving LC3B affinity and selectivity. These peptides represent the highest‐affinity LC3B‐selective ligands reported to date, and they will be useful tools for further elucidation of LC3B's role in autophagy and in cancer.