In vitro neuronal models are valuable tools for studying neural diseases and neurotoxicology. With the use of microelectrode arrays, the Maestro Pro can measure the electrical activity of neural cultures in a high throughput, label-free manner. Here, an optimized in vitro assay is demonstrated to characterize the response of neural cultures to neuroactive compounds. 40,000 primary cortical rat neurons per well were plated on CytoView 24-well plates, and neural activity and viability were monitored on the Maestro Pro. To determine the optimal culture conditions for compound dosing, neural network activity, synchrony, and oscillation were assessed at DIV14 of neural cultures in three different media systems. BrainPhys + SM1 was the optimal choice for this application because it induced a moderate level of baseline neural activity, allowing for the detection of increases or decreases in all three measures following compound dosing. After optimizing neural culture conditions for dosing, recordings taken on the Maestro Pro revealed that 4-aminopyrirdine led to increases in activity, synchrony, and oscillation. In contrast, glutamate significantly increased activity but decreased synchrony and oscillations. Picrotoxin increased synchrony but did not affect oscillatory behavior, while carbachol decreased synchrony slightly but significantly increased oscillations. Viability did not significantly change following dosing, indicating that changes in neural activity were not due to cell death. In total, this study shows that the Maestro Pro is a valuable tool for optimizing in vitro neural models and detecting electrophysiological changes from neuroactive compounds.