As key players in cell function, ion channels are important targets for drug discovery and therapeutic development against a wide range of health conditions. Thus, developing assays to reconstitute ion channel macromolecular complexes in physiological conditions and screen for chemical modifiers of protein–protein interactions within these complexes is timely in drug discovery campaigns. For most ion channels, expressing their pore-forming subunit in heterologous mammalian cells has now become a routine procedure. However, reconstituting protein-channel complexes in physiological environments is still challenging, limiting our ability to identify tools and probes based on allosteric mechanisms, which could lead to more targeted and precise modulation of the channel function. Here, we describe the assay development steps to stably reconstitute the interaction between voltage-gated Na+ (Nav) channel Nav1.6 and its accessory protein, fibroblast growth factor 14 (FGF14) using the split-luciferase complementation assay (LCA), followed by assay miniaturization and optimization in 384-well plates for in-cell high-throughput screening (HTS) against protein-channel interactions. This optimized LCA can subsequently be used for rapid estimation of hit potency and efficacy via dose-dependency studies, enabling ranking of hits prior to more labor-intensive validation studies. Lastly, we introduce the methodology for rapid functional hit validation studies using semi-automated planar patch-clamp electrophysiology. Our robust, in-cell HTS platform can be adapted to any suitable ion channel complex to explore regulatory pathways of cellular signaling using kinase inhibitors, as well as to screen small molecules for probe development and drug repurposing toward new targets/areas of medicine. Overall, the flexibility of this assay allows users to broadly explore therapeutic options for channelopathy-associated diseases at a fast pace, enabling rapid hypothesis generation in early phase drug discovery campaigns and narrowing down targets prior to more labor-intensive in vivo studies.