Peptide toxins folded into an inhibitor cystine knot (ICK) are found in the venoms of a variety of organisms (spider, scorpion, snakes, etc.), and many of these target the voltage-sensing domains in voltage-activated ion channels to modulate their gating properties. Our previous studies suggest that tarantula toxins partition into the lipid membrane and bind to the S3b-S4 paddle motif within the voltage-sensing domains to allosterically inhibit Kv channels. Alanine scanning mutagenesis studies have identified candidate residues in both toxin and channel that may contribute to forming the protein-protein interface when in complex. To definitively identify protein interaction surfaces between toxin and Kv channel, we employed unnatural amino acids and Strain - Promoted Azide Alkyne Click (SPAAC) chemistry to carry out site-specific crosslinking. Azidohomoalanine (AHA), an analog of methionine, was incorporated in Kv channels expressed in Xenopus laevis oocytes through endogenous methionyl t-RNA synthetase by supplementing excess of AHA in the medium. Simultaneously, guangxitoxin-1E (GxTx-1E) was engineered with spinster cysteine residues to incorporate an azide-reactive dibenzocyclooctyne (DBCO) group using cysteine-maleimide conjugation method. Currently, we are assaying for toxin-channel crosslinks in a functional manner by analyzing the recovery of macroscopic ionic currents and kinetics of toxin dissociation using two-electrode voltage clamp (TEVC) technique.