Native chemical ligation followed by desulfurization is a powerful strategy for the assembly of proteins. Here we describe the development of a highyielding, one-pot ligation desulfurization protocol that uses trifluoroethanethiol (TFET) as a novel thiol additive. The synthetic utility of this TFET-enabled methodology is demonstrated by the efficient multi-step one-pot syntheses of two tick-derived proteins, chimadanin and madanin-1, without the need for any intermediary purification.