With an emphasis on our studies focused on 3-nitro-2-pyridinesulfenyl (Npys) unit,1,2 we report herein a new “two step solid-phase disulfide ligation” method. This is a strategy to prepare a disulfide peptide from two different peptide fragments containing cysteine residues.3 Briefly, a peptide fragment A containing Cys(t-Bu) is loaded onto Npys chloride resin via the formation of an active disulfide bond. The resulting resin-attached peptide A is then readily transferred to another Cys-containing peptide fragment B by a disulfide exchange reaction, selectively forming and releasing the new disulfide peptide C from the resin. Peptide C is easily recovered in high purity by filtration. Subsequent amide bond formation yields the desired cyclic peptide. This strategy sets up a novel disulfide-led synthetic methodology for disulfide-containing cyclic peptides. During the presentation, the syntheses of cyclic peptides, oxytocin and endothelin will be discussed as a fundamental model towards more complex cyclic peptides. As another application, since a notable difference in solubility between hydrophilic biocompatible carriers and hydrophobic small-molecule drugs makes their chemical conjugation difficult, particularly in peptide–drug conjugation, we adopted the above-mentioned two-step solid-phase disulfide ligation.4 Suitable solvent systems can be used for reactant solubilization during each step. Hydrophobic antitumor agent plinabulin5 and hydrophilic octaarginine were successfully conjugated via disulfide bond as a practical model.