The human Central Nervous System (CNS) has a unique structural organization that is critical to its complex functions. Efforts to model this intricate network in vitro have encountered major bottlenecks. Recently, much work has been focused on obtaining 3D brain organoids in an attempt to better recapitulate the brain development and function in vitro. Although self-organized 3D organoids can potentially more closely recapitulate key features of the human CNS, current protocols still need major improvements before being implemented in a drug discovery scenario. We have recently described a highly homogenous off-the-shelf human induced Pluripotent Stem Cells (hiPSCs)-derived cortical spheroid screening platform in 384 well format, composed of cortical neurons and astrocytes. Using high throughput calcium flux analysis, we showed the presence of quantifiable, robust and uniform spontaneous calcium oscillations, which is correlated with synchronous neuronal activity in the spheroid. Our platform is optimized to have a highly homogenous and consistent functional signal across wells, plates, and batches. Finally, we demonstrated the feasibility of using this platform to interrogate large libraries of compounds on their ability to modulate the human CNS activity. Here, we describe the use of this platform to investigate neurodevelopmental disorders. When introducing hiPSCs derived from Rett Syndrome (RTT) patients into our platform, a clear functional disease phenotype was observed. RTT 3D neural cultures displayed calcium signal that indicates a compromised neural network with slow, large, synchronized frequency of oscillations. We also performed a pilot screen using a library of 296 selected compounds for their ability to alleviate the observed RTT phenotypes in vitro, and identified some potential targets. In summary, we demonstrated the feasibility of incorporating a neurodevelopmental disorder in a high-throughput screening platform. The system presented here has the potential to dramatically change the current drug discovery paradigm for neurodevelopmental disorders and other neural diseases.