Insulin is the principal hormone involved in the regulation of metabolism and has served a seminal role in the treatment of diabetes. While a miraculous substance, insulin has notable limitations as a medicine and consequently the quest for safer and more convenient therapy continues. We explored a comprehensive disulfide scan in search of a single additional disulfide bond that could regulate bioactivity in a binary manner, presumably by reducing conformational flexibility. Building upon recent advances in insulin synthetic methodology we have developed a straight-forward route to preparation of nearly two dozen novel analogs that employed a unique [3+1] strategy. Subsequent to formation of the three disulfide bonds in native insulin, an additional fourth site-specific disulfide was selectively formed from two S-Acm protected cysteines. The bioactivity was established for the constrained (4-DS) and unconstrained (3-DS) analogs by in vitro methods, and extended to in vivo study for select peptides. All the 4-DS peptides (n=19) were successfully synthesized with minor differences in yield, regardless of the projected distance separating the cysteines constituting the fourth disulfide. Conversely, with only a single exception, the attempt to simultaneously form all four disulfide bonds was uniformly unsuccessful. These results demonstrate an unforeseen ability of native insulin to structurally accommodate an additional covalent tether when properly introduced by a two-step synthesis. We also identified a preferred anchor point where a single disulfide bond can significantly regulate insulin activity. We believe that the described [3+1] methodology might constitute the preferred approach for performing similar disulfide scanning in peptides that contain multiple disulfides.