Neural 3D organoids from human induced pluripotent stem cells (iPSC) are a rapidly developing technology with great potential for understanding brain development and neuronal diseases. A promising parallel approach is assembling structures similar to 3D spheroids or “neurospheres” by using defined combinations of fully differentiated human iPSC-derived cells in tri-culture, including glutamatergic neurons, GABAergic neurons, and astrocytes. In this study, we chose to model Alzheimer’s Disease (AD) by incorporating allelic variants of the ApoE gene (2/2, 3/4, and 4/4) to create disease-specific “neurospheres”. 3D spheroids were formed by combining 20-25,000 cells in ultra-low attachment (ULA) plates. We tracked spheroid morphology over time by live cell imaging. Microtissues were also analyzed by confocal fluorescence imaging for cell organization and expression of various neural markers, including TUJ1 and GFAP. Functional performance was tested via calcium oscillation assay and was run on a FLIPR instrument capable of fast kinetic recordings. The calcium oscillation patterns were analyzed for metrics like peak count, amplitude, and width. For pharmacological characterization, a panel of select compounds was used to show both the appropriate responses to GABA, AMPA, and NMDA, as well as changes to neuroactive compounds. Some drugs previously shown to affect AD phenotypes (memantine and donepezil) decreased calcium peak amplitude and altered other metrics as visualized by ScreenWorks software and analyzed by PeakPro2. This biological system of 3D neurospheres assembled from human iPSC-derived cell types paired with high-content imaging and detailed analysis of calcium oscillations demonstrates a promising tool for disease modeling and compound testing.