Cyclic peptides have been receiving increasing attention as potential therapeutic agents. Cyclisation can improve essential properties such as selectivity, binding affinity, and bioavailability. In addition, cyclisation can also enhance metabolic stability allowing for the design of enzyme inhibitors that resist proteolytic digestion. The current work involves the synthesis of inhibitors through a novel two-component peptide cyclisation method, capitalising on a pyridine-2,6-dicarbonitrile reagent that reacts with two 1,2-aminothiol moieties in a peptide. Two strategies for the introduction of the 1,2-aminothiol motif into peptides are demonstrated. The motif can either be resembled by canonical N-terminal cysteine in the peptide main chain or by a new unnatural amino acid that displays an N-terminal cysteine in the peptide side chain. The approach is fully compatible with standard Fmoc solid-phase peptide synthesis and the linear peptide can be isolated using HPLC purification. Cyclisation is performed in an aqueous buffer at pH 7.5 and typically completes in less than one hour. We have demonstrated this approach for the cyclisation of peptide substrates of viral drug targets. We incorporated the novel unnatural amino acid at two positions of the substrate sequence to produce a high-affinity cyclic inhibitor of the Zika virus protease. In a second example, we placed the unnatural amino acid at the C-terminus and a canonical cysteine residue at the N-terminus of the Chikungunya virus protease substrate. The novel two-component approach described in this study has proven to be a very simple, effective, and biocompatible tool for peptide cyclisation with obvious future applications in peptide stapling technologies.