A functional, membrane potential FRET-based assay for Nav1.7 channels. In the absence of other ionic conductances that can hyperpolarize the cell, heterologous expression of Nav1.7 channels provides a system where at the cell resting membrane potential most channels will reside in the nonconducting inactivated state. Removal of fast inactivation by the addition of veratridine shifts the channel's equilibrium to the conductive, open state that allows sodium entry leading to cell depolarization. The changes in voltage can be monitored with a pair of FRET voltage-sensing dyes, coumarin and oxonol. Cell depolarization alters the distribution of oxonol across the membrane, causing a change in the FRET signal. In the presence of a Nav1.7 inhibitor, channel equilibrium shifts toward the inactivated, drug-bound conformation, reducing the number of channels that will be available for veratridine modification, and preventing the agonist-induced FRET signal. The dose–response curve for the veratridine-induced change in FRET signal is steep, suggesting that modification of a small number of Nav1.7 channels is sufficient to cause cell depolarization.