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In vitro neuronal models are valuable tools for studying brain function, neural diseases, and toxicology. These models often consist of a 2D monolayer of cells that are used to screen the neural effects of compounds of interest. However, recent advances in induced pluripotent stem cell (iPSC) technology have allowed researchers to produce 3D models of neural tissue, termed neural organoids, that better recapitulate the cellular diversity and spatial architecture of in vivo tissue. Here, a toxicology screening workflow to monitor the differentiation of neural organoids and their response to neuroactive compounds is described. Neural organoids were both generated via commercially available kits and purchased as a finished product for this study. To generate neural organoids, iPSCs were first plated and monitored using a whole-vessel imaging system (the Omni). Colony area, diameter, and coverage increased over a three-day period, at which point the colonies reached an optimal size for passaging and subsequent embryoid body formation. Following neural induction using STEMCELL Technologies’ Dorsal Forebrain Organoid Differentiation Kit (Catalog #08620), organoids in suspension culture were imaged for 50+ days using the Omni, and organoid size and count were monitored, illustrating the progression of the differentiation process. For electrophysiological measurements, pre-formed Human iPSC-Derived Midbrain Organoids (Catalog #200-0792 and Catalog # 200-0793) from STEMCELL Technologies were cultured using the STEMdiffTM Neural Organoid Maintenance Kit (Catalog #100-0120). The midbrain organoids were plated onto CytoView 6 MEA plates and, on Day 125 post-differentiation, dosed with neuroactive compounds or a DMSO control. Baseline and postdose (1 hour after drug addition) recordings were taken using the Maestro Pro and showed robust changes in midbrain organoid activity patterns in response to the potassium channel blocker 4-aminopyrirdine and in response to the mitochondrial complex I inhibitor rotenone. Dosing with 4-aminopyrirdine increased the mean firing rate and network burst frequency of midbrain organoids, while dosing with rotenone led to a decrease in both metrics. Importantly, neither compound caused decreases in resistance measurements, signaling that network activity changes were due to electrophysiological changes rather than cell death. In total, this study shows that the Omni and Maestro Pro systems are valuable tools for studying the differentiation of neural organoids and their subsequent use in toxicological screening applications. Approaches using these organoid workflows can produce data at the functional level that may more closely mimic in vivo physiology and better inform the use of compounds in the clinic.