Abstract: Neural 3D spheroids are a rapidly developing technology with great potential for understanding brain development and neuronal diseases. They provide a more advanced and biologically relevant system for basic research and high-throughput drug discovery, including compound profiling and toxicity testing. Here, we describe methods for assembling human iPSC-derived cell types, including glutamatergic neurons, GABAergic neurons, and astrocytes into 3D neurospheres. For disease modelling of epilepsy phenotypes, we used two different genetically modified GABAergic neurons (SCN1A KO or KCNT1 P924L mutation) and their isogenic pairs as matched controls. The SCN1A gene encodes the alpha subunit of the sodium channel NaV1.1 and it is the major gene implicated in Dravet Syndrome, a severe childhood epilepsy. The KCNT1 gene encodes a potassium channel and the P924L mutation is linked to an early-onset epileptic encephalopathy. We monitored the formation, morphology, and functional activity (Ca2+ oscillations) of the neurospheres after 3 weeks in culture. The microtissues were also analyzed by confocal fluorescence imaging for cell organization and expression of neuronal markers (TUJ1) and astrocytes (GFAP). Cellular and spheroid morphology was characterized by using high-content imaging. The calcium assay was performed on a FLIPR Penta instrument capable of fast kinetic recordings using a calcium-sensitive dye and oscillation patterns were analyzed for peak frequency, amplitude, width, & spacing. Different baseline oscillation patterns were observed between control and disease neurospheres, however within each group calcium kinetics and patterns were highly consistent. For pharmacological characterization of the control and diseased phenotypes, we used a panel of 14 compounds, including selected molecules that affect GABA, AMPA, NMDA, sodium and potassium channels, dopamine receptors, as well as select neuroactive and neurotoxic substances. The functional responses demonstrated the predicted effects based on mode of action, consistent across control and disease model 3D neurospheres. Notably, moderately increased excitability was observed for mutated phenotypes, showing in both, in the baseline pattern, also in the elevated responses to stimulating agents. This biological system of 3D neurospheres paired with high-content imaging and detailed analysis of calcium oscillations demonstrates a promising tool for disease modeling and compound testing.