The article presents a novel approach to the discovery of mirror-image D-peptide ligands using automated flow peptide synthesis (AFPS) and mirror-image phage display (MIPD). The authors demonstrate a general pipeline that enables the rapid preparation of D-proteins in a single run, overcoming the challenges associated with individualized optimization required for D-protein synthesis. By using AFPS, they successfully prepared and characterized 12 D-proteins, which is almost one-third of all reported D-proteins to date. These D-proteins were then used in MIPD to discover six macrocyclic D-peptide binders: three to the oncoprotein MDM2 and three to the E3 ubiquitin ligase CHIP. The successful production of mirror-image proteins opens up new avenues for D-peptide drug discovery and the study of mirror-image biology. The article highlights the advantages of D-peptides, such as their metabolic stability and resistance to degradation, and discusses the limitations of traditional peptide synthesis methods. The use of AFPS and MIPD together provides a robust and scalable platform for generating mirror-image ligands, which could significantly advance the field of peptide-based therapeutics.The article presents a novel approach to the discovery of mirror-image D-peptide ligands using automated flow peptide synthesis (AFPS) and mirror-image phage display (MIPD). The authors demonstrate a general pipeline that enables the rapid preparation of D-proteins in a single run, overcoming the challenges associated with individualized optimization required for D-protein synthesis. By using AFPS, they successfully prepared and characterized 12 D-proteins, which is almost one-third of all reported D-proteins to date. These D-proteins were then used in MIPD to discover six macrocyclic D-peptide binders: three to the oncoprotein MDM2 and three to the E3 ubiquitin ligase CHIP. The successful production of mirror-image proteins opens up new avenues for D-peptide drug discovery and the study of mirror-image biology. The article highlights the advantages of D-peptides, such as their metabolic stability and resistance to degradation, and discusses the limitations of traditional peptide synthesis methods. The use of AFPS and MIPD together provides a robust and scalable platform for generating mirror-image ligands, which could significantly advance the field of peptide-based therapeutics.