The ligation of sterically demanding peptidyl sites such as those involving Val-Val and Val-Pro linkages has proven to be extremely challenging with conventional NCL methods that rely on exogenous thiol additives. Herein, we report an efficient β-thiolactone-mediated additive-free NCL protocol that enables the establishment of these connections in good yield. The rapid NCL was followed by in situ desulfurization. Reaction rates between β-thiolactones and conventional thioesters towards NCL were also investigated, and direct aminolysis was ruled out as a possible pathway. Finally, the potent cytotoxic cyclic-peptide axinastatin 1 has been prepared using the developed methodology.