The purpose of this study was to develop and optimize a unique one-pot, two-step site-specific PEGylation method suitable for the high-yield production of mono-PEGylated (Lys(18)) salmon calcitonin (Lys(18)-PEG-sCT), which was previously demonstrated to have superior pharmaceutical properties to other conjugates. For the site-specific PEGylation, this study used the sCT derivative (FMOC(1,11)-sCT), which was FMOC protected at Cys(1)- and Lys(11)-amines among three PEGylation sites including Lys(18)-amine. This PEGylation process was achieved by the consecutive one-pot, two-step reaction: (i) the PEG conjugation to FMOC(1,11)-sCT; and (ii) the subsequent deprotection of FMOC group from the PEGylated FMOC(1,11)-sCT. The optimized reaction resulted in the high production yield of Lys(18)-PEG-sCT (about 86%), compared with that from conventional non-specific PEGylation (about 18%). The prepared Lys(18)-PEG-sCT conjugate showed improved biological stability without the loss in the in vitro and in vivo biological activity by PEGylation. Consequently, this site-specific PEGylation using an FMOC protection/deprotection strategy showed great usefulness in the production of the most promising Lys(18)-PEG-sCT conjugate with a high yield.