ML308 was identified as a novel inhibitor of the potassium channel, subfamily K, member 9 (KCNK9) two-pore domain potassium channel. Two-pore domain potassium channels provide a background leak conductance that is selectively permeable to potassium. These channels regulate cell membrane potential and excitability and thereby modulate a variety of processes including hormone secretion, proliferation, and central nervous system (CNS) function. A high throughput fluorescent screen measuring thallium influx through KCNK9 channels was used to identify bisamide and thiotriazole classes of inhibitors. Chemical modification of the thiotriazole scaffold yielded ML308 which displayed a potent block of KCNK9 channels in a thallium influx fluorescent assay (IC50 = 130 nM) and in an automated electrophysiology assay (IC50 = 413 nM). ML308 afforded >50-fold selectivity for block of KCNK9 over the closely-related, two-pore domain potassium channel, KCNK3, in fluorescent assays and displayed little or no block at 10 μM of the more distantly related potassium channels, Kir2.1, potassium voltage-gated channel, KQT-like subfamily, member 2 (KCNQ2), and human ether-a go-go-related gene (HERG). The potency and selectivity profile of ML308 makes it a useful pharmacological probe for in vitro studies of KCNK9 function and in further studies aimed at therapeutic intervention.