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Our research aimed to assess the replacement of HF with Trifluoromethanesulfonic acid (TFMSA). Here we examined a 33 amino acid test peptide, Huwentoxin\u2010I (HwTx\u2010I) as a novel candidate for our TFMSA cleavage protocol. Structurally HwTx\u2010I has an X\u2010Cys<jats:sup>16<\/jats:sup>\u2010Cys<jats:sup>17<\/jats:sup>\u2010X sequence mid\u2010region, which makes it an ideal candidate for NCL. Experiments determined that the best yields (16.8%) obtained for 50 mg of a thioester support resin were achieved with a TFMSA volume of 100 \u03bcL with a 0.5\u2010h incubation on ice, followed by 2.0 h at room temperature. RP\u2010HPLC\/UV and mass spectra indicated the appropriate parent mass and retention of the cleaved HwTx\u2010I <jats:italic>N\u2010<\/jats:italic>terminal thioester fragment (Ala<jats:sup>1<\/jats:sup>\u2010Cys<jats:sup>16<\/jats:sup>), which was used in preparation for NCL. The resulting chemically ligated HwTx\u2010I was oxidized\/folded, purified, and then assessed for pharmacological target selectivity. Native\u2010like HwTx\u2010I produced by this method yielded an EC<jats:sub>50<\/jats:sub> value of 340.5\u2009\u00b1\u200926.8 nM for Na<jats:sub>v<\/jats:sub>1.2 and an EC<jats:sub>50<\/jats:sub> value of 504.1\u2009\u00b1\u200981.3 nM for Na<jats:sub>v<\/jats:sub>1.3, this being similar to previous literature results using native material. This article represents the first NCL based synthesis of this potent sodium channel blocker. Our illustrated approach removes potential restrictions in the advancement of NCL as a common peptide laboratory technique with minimal investment, and removes the hazards associated with HF usage. \u00a9 2016 Wiley Periodicals, Inc. 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