Summary Heart disease has been the #1 cause of fatality in the United States since 1950. Additionally, cardiovascular safety liability especially pro-arrhythmic risk remains a leading cause for the high attrition rate in drug development. For investigation of mechanisms and potential therapies for cardiac dysfunction, there has been strong demand from industry and academic alike for high quality, high-throughput assay of cardiac action potential (AP), a powerful biomarker for cardiac dysfunction. Unfortunately, existing techniques for measurement of AP, including electrical and optical methods, all suffer from severe limitations and are technically challenging and/or costly to execute at scale. These limitations are further compounded by the profound heterogeneity of human-derived cardiomyocytes. This Phase I proposal aims to develop a novel in vitro electrophysiology system that supports high-throughput measurement of intracellular AP (iAP) using human-derived cardiomyocytes. More specifically, we seek to demonstrate the feasibility for researcher even with limited expertise in electrophysiology to perform high-throughput iAP assay that includes data acquisition and analysis. Central to this electrophysiology system is a 3D nanoelectrode array (NEA) developed at Stanford University, which bridges the gap between intracellular and extracellular electrophysiology and enables high-quality and scalable recordings of iAP from many individual cardiomyocytes. Based on this NEA technology, Cyion Technologies has previously developed a first-generation electrophysiology system (Vincent) that records iAP signals from up to 60 discrete cardiomyocytes in a single-well NEA substrate. Built on this success, in this proposal we seek to develop an additional “plate-reader” option that measures 672 individual cardiomyocytes using a 96-well NEA substrate. We will also implement a software algorithm in user software for automated, batch analysis of iAP signals, which is necessary for higher-throughput assay.