The enteric nervous system (ENS) is a complex neuronal network organized in ganglionated plexuses that extend along the entire length of the gastrointestinal tract. Largely independent of the central nervous system, the ENS coordinates motility and peristalsis of the digestive tract, regulates secretion and absorption, and is involved in immunological processes. Electrophysiological methods such as the patch-clamp technique are particularly suitable to study the function of neurons as well as the biophysical parameters of the underlying ion channels under both physiological and pathophysiological conditions. However, application of the patch-clamp method to ENS neurons remained difficult because they are embedded in substantial tissue layers that limit access to and targeted manipulation of these cells. Here, we present a robust step-by-step protocol that involves isolation of ENS neurons from adult mice, culturing of the cells, their transfection with plasmid DNA, and subsequent electrophysiological characterization of individual neurons in current-clamp and voltage-clamp recordings. With this protocol, ENS neurons can be prepared, transfected, and electrophysiologically characterized within 72 h. Using isolated ENS neurons, we demonstrate the feasibility of the approach by functional overexpression of recombinant voltage-gated NaV1.9 mutant channels associated with hereditary sensory and autonomic neuropathy type 7 (HSAN-7), a disorder characterized by congenital analgesia and severe constipation that can require parenteral nutrition. Although our focus is on the electrophysiological evaluation of isolated ENS neurons, the presented methodology is also useful to analyze molecules other than sodium channels or to apply alternative downstream assays including calcium imaging, proteomic and nucleic acid approaches, or immunochemistry.