Disulfide-rich peptides are promising therapeutic and diagnostic agents, but their preparation is often limited by the tedious and low-yielding folding process. We found that a single cystine-to-diaminodiacid displacement could significantly increase the folding efficiency of disulfide-rich peptides and therefore, greatly improve their production yields. The practicality of this strategy was demonstrated by the efficient synthesis and folding of the derivatives of a m-conotoxin SIIIA, a preclinical hormone hepcidin, and a trypsin inhibitor EETI-II. NMR and X-ray crystal structure studies confirmed that these disulfide-rich peptide derivatives maintained correct three-dimensional conformations. Moreover, the cystine-to-diaminodiacid displacement enabled structural tuning at a new dimension, leading to an EETI-II derivative with higher bioactivity than the native peptide.