KCNQ voltage-gated potassium channels are involved in regulating excitability, action potential duration and transport processes in a variety of cell types. A high throughput fluorescent screen based depolarization-triggered thallium influx through KCNQ1 channels was developed and used to screen the Molecular Libraries Probe Production Centers Network (MLPCN) library to identify activators of KCNQ1 channels. Identified hits were characterized using IonWorks automated electrophysiology. An initial racemic hit (EC50 = 1.7 μM) served as a starting point for optimization studies of newly synthesized compounds. ML277 was identified as a potent (EC50 = 0.26 μM) activator of KCNQ1 channels with more than 100-fold selectivity for activation of KCNQ1 channels compared with closely related KCNQ2 and KCNQ4 channels and with distantly related hERG potassium channels. ML277 provides a novel and selective chemical probe to investigate the role of KCNQ1 channels.