Mirror-image biological systems have the potential for broad-reaching impact in health and diagnostics, but their study has been greatly limited by the lack of routine access to synthetic D-proteins. We demonstrate that automated fast flow peptide synthesis (AFPS) can reliably produce novel mirror-image protein targets without prior sequence engineering. We synthesized 12 D-proteins, along with their L-counterparts. All 24 synthetic proteins were folded into active structures in vitro, and characterized using biochemical and biophysical techniques. From these chiral protein pairs, we chose MDM2 and CHIP to carry forward into mirror-image phage display screens, and identified macrocyclic D-peptides that bind the recombinant targets. We report 6 mirror-image peptide ligands with unique binding modes: three to MDM2, and three to CHIP, each confirmed with X-ray co-crystal structures. Reliable production of mirror-image proteins with AFPS stands to enable not only the discovery of D-peptide drug leads, but to the study of mirror-image biological systems more broadly.