Although heart failure is the leading cause of death in the U.S., therapeutic treatments remain suboptimal as the pharmaceutical industry has proven incapable of generating more predictive human-relevant preclinical models of the human heart that are amenable to high-throughput screening (HTS) assays. Thus, vast compound libraries remain virtually unexplored in an effective manner, hindering the speedy development of new therapeutic approaches to treat heart disease. Organos, Inc. proposes a solution to this problem in the commercial development of an in vitro miniaturized array of “Micro-Heart Muscle” (µHM) amenable to HTS. Organos’ innovation is that our HTS capable assay is based on heart muscle contraction metrics, which to date has neither been achieved nor is attainable in two-dimensional (2D) platforms. A main focus of this proposal is to create an HTS assay, using µHMs that fit a 384 microplate form factor, amenable to use with pre-existing automation. In the µHM, elongated muscle fibers are formed from induced pluripotent stem cell-derived cardiomyocytes (hiPSC- CMs) in polymer templates that promote uniaxial alignment and contraction, robust sarcomere assembly, and physiologically relevant drug responsiveness. The µHM 384 HTS platform and assay will lead the front end of Organos’ discovery pipeline and feed data into our computational and deep learning models for compound discovery, target identification, and repurposing. The Specific Aims focus first on the fabrication of the HTS µHM platform, microtissue characterization, and assay development, then on validation and its use in HTS to identify compounds that improve cardiac contractility. The main outcome will be a robust HTS assay employing human heart muscle suitable for use in large screening campaigns for de novo drug discovery.