Venom peptides are an excellent source of pharmacologically active molecules for ion channels that have been considered as promising drug targets. However, mining venoms that interact with ion channel remains challenging. Previously an autocrine based high throughput selection system was developed to screen venom peptide library but the method includes repetitious selection rounds that may cause loss of valuable hits. To simplify the selection process, next generation sequencing was employed to directly identify the positive hits after a single round of selection. The advantage of the improved system was demonstrated by the discovery of 3 novel Kv1.3 targeting venom peptides among which Kappa-thalatoxin-Tas2a is a potent Kv1.3 antagonist. Therefore, this simplified method is efficient to identify novel venom peptides that target ion channels.