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D-peptides are recognized as a new class of synthetic chemical drugs and they possess many interesting advantages such as high enzymatic stability, improved oral bioavailability, as well as high binding affinity and specificity. Recently, D-peptide drugs have been attracting increasing attention in both academic and industrial researches over recent years. One D-peptide etelcalcetide has even entered the market that targets the calcium (Ca2+)-sensing receptor (CaSR) to fight secondary hyperparathyroidism. Effective discovery and optimization of D-peptide ligands that can bind to various disease-related targets with high specificity and potency is of great importance for the development of D-peptide drugs. This review surveys the recent method development in this area especially the chemical protein synthesis-assisted high-throughput screening strategies for D-peptide ligands and their application in drug discovery.