Voltage-gated sodium (NaVs) channels are pore-forming transmembrane proteins that regulate the influx of sodium ions across cell membranes. Spider venoms are a rich source of NaV-modulating peptides with high selectivity and potency, making them important tools for understanding NaV structure and function. NaV1.8 is expressed in the peripheral nervous system and contributes to the propagation of action potentials in nociceptive neurons, making it a potential therapeutic target for pain. To date, only a few venom-derived peptides with subnanomolar potency at NaV1.8 have been described. Therefore, the aim of this study was to assess the potency, selectivity, and mechanism of action of Tsp2a, a 36 amino acid residue peptide isolated from the crude venom of Thrixopelma sp., which was identified from a high-throughput screen as an inhibitor of NaV1.8. Tps2a was synthesized using solid-phase peptide synthesis and activity was assessed using automated whole-cell patch-clamp recordings (QPatch-16 and QPatch Compact) in HEK293 or CHO cells expressing NaV1.4, NaV1.5, NaV1.7, NaV1.8 and KV2.1. Tsp2a inhibited NaV1.8 peak current (IC50 210 nM) while causing a depolarising shift in the voltage dependence of activation (ΔV1/2=11.30 ± 0.8 mV) and a small persistent current. Tsp2a inhibited peak current with similar potency at NaV1.5 (IC50 282 nM) and KV2.1 (IC50 156 nM), and was 10-fold selective over NaV1.4 (IC50 1769 nM) and NaV1.7 (IC50 1278 nM). We next synthesized Tsp2a analogues to understand the structure-activity relationship that gives rise to activity at TTX-resistant NaV channels. The results of this work will help identify key residues on spider-venom derived peptides involved in NaV1.8 channel binding for the future rational design of selective NaV1.8 modulators.