The human ether à go-go 1 (hEag1) ion channel, also known as Kv10.1, is a voltage-gated potassium channel that is highly expressed in the brain but is almost completely absent in other tissues under healthy conditions. However, during cancer development, hEag1 is upregulated in various tumors, making it a biomarker and therapeutic target. Its role in cancer remains poorly understood, making hEag1 an interesting target for study. However, no ligands that selectively and potently bind hEag1 are currently available. In this work, we developed a series of selective hEag1 ligands that can serve as pharmacological probes and therapeutic leads to investigate the channel's function in breast cancer. We leveraged the rich pharmacological diversity of animal venoms to explore peptide ligands targeting hEag1. Through structure-activity relationship studies, the potency and selectivity of two venom-derived peptides, APETx4 and Aa1a, were studied. APETx4 was characterized from the venom of the sea anemone Anthopleura elegantissima, while Aa1a was isolated from the spider venom of Avicularia aurantiaca. These peptides and their analogs were tested for activity on Kv10.1 expressed in Xenopus laevis oocytes, using the two-electrode voltage-clamp technique. The results show that residues important for activity could be identified. The binding site of these peptides on Kv10.1 was also investigated using the scanning cysteine accessibility mutagenesis method. By combining peptide chemistry and pharmacology, this research paves the way for the creation of highly specific hEag1-targeting probes. The optimized compounds will be used in future studies to explore the functional role of hEag1 in breast cancer progression and its therapeutic potential. The aim is to improve the understanding of hEag1’s role in cancer and unlock new avenues for targeted cancer therapy.