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Hi1a is a venom peptide isolated from the Australian funnel-web spider , containing an intricate "double knot" tertiary structure, and is currently under investigation for the treatment of ischemic stroke. The recombinant expression and chemical synthesis of Hi1a remain a significant challenge due to laborious protocols, delaying progress in translation to the clinic. We describe the first single-shot chemical synthesis of Hi1a (76 amino acids, AA) using automated fast-flow peptide synthesis (AFPS), enabling rapid access (<4.5 h total synthesis time) to milligram quantities of linear Hi1a (>10 mg). This work highlights the utility of AFPS as a technology that enables chemical production of biomolecules challenging to obtain by recombinant methods.