Human induced pluripotent stem cell (hiPSC)-based three dimensional (3D) platforms enable greater physiological relevance, improving performance in drug discovery and toxicology applications. These complex, biologically relevant, and predictive cell-based assays are ideal for rapidly screening environmental toxins and chemical compounds. In this Innovation Showcase, we will describe how we used the StemoniX microBrain 3D platform, a high throughput screening system comprised of human iPSC-derived homogenous 3D neural spheroids, to screen a diverse 91 compound-library from the National Toxicology Program. Each uniform spheroid has a fully developed network of cortical neurons and astrocytes co-matured from a single donor source. Immunocytochemistry indicates that the neurons and astrocytes display key markers of cellular identity and maturity, such as synaptic proteins and glutamate transporters. With its fully functional glutamatergic and GABAergic circuitry, microBrain 3D spheroids present highly synchronized, quantifiable, robust, and uniform spontaneous calcium oscillations that can be easily reproducibly modified and screened using fluorometric imaging, kinetic high throughput readers plate readers such as the FLIPR Tetra® High-Throughput Cellular Screening System, and, at the cellular level with high-content imaging systems such as the ImageXpress® Micro Confocal High-Content Imaging System.