A new method for rapidly synthesizing D-proteins using automated flow peptide synthesis (AFPS) has enabled the discovery of mirror-image D-peptide ligands for biological targets. This approach allows for the efficient production of D-proteins, which are chiral counterparts of L-proteins, and facilitates the use of mirror-image phage display (MIPD) to identify high-affinity D-peptide binders. The study demonstrates the synthesis of 12 D-proteins, including those that have not been previously synthesized, and the discovery of six macrocyclic D-peptide binders to two protein targets: MDM2 and CHIP. These D-peptides show similar binding affinities to their L-protein counterparts, indicating that the synthetic D-proteins are biologically active. The results highlight the potential of D-peptides as therapeutic scaffolds and demonstrate the feasibility of using AFPS to overcome the challenges associated with the synthesis of mirror-image proteins. The study also shows that mirror-image D-peptides can be used to probe biological systems and may have applications in drug discovery and the study of mirror-image biology. The findings suggest that the combination of AFPS and MIPD can significantly enhance the throughput and accessibility of D-peptide binder discovery.A new method for rapidly synthesizing D-proteins using automated flow peptide synthesis (AFPS) has enabled the discovery of mirror-image D-peptide ligands for biological targets. This approach allows for the efficient production of D-proteins, which are chiral counterparts of L-proteins, and facilitates the use of mirror-image phage display (MIPD) to identify high-affinity D-peptide binders. The study demonstrates the synthesis of 12 D-proteins, including those that have not been previously synthesized, and the discovery of six macrocyclic D-peptide binders to two protein targets: MDM2 and CHIP. These D-peptides show similar binding affinities to their L-protein counterparts, indicating that the synthetic D-proteins are biologically active. The results highlight the potential of D-peptides as therapeutic scaffolds and demonstrate the feasibility of using AFPS to overcome the challenges associated with the synthesis of mirror-image proteins. The study also shows that mirror-image D-peptides can be used to probe biological systems and may have applications in drug discovery and the study of mirror-image biology. The findings suggest that the combination of AFPS and MIPD can significantly enhance the throughput and accessibility of D-peptide binder discovery.