Three-dimensional neuronal culture systems such as spheroids, organoids, and assembloids constitute a branch of neuronal tissue engineering that has improved our ability to model the human brain in the laboratory. However, the more elaborate the brain model, the more difficult it becomes to study functional properties such as electrical activity at the neuronal level, similar to the challenges of studying neurophysiology in vivo. We describe a simple approach to generate self-assembled three-dimensional neuronal spheroid networks with defined human cell composition on microelectrode arrays. Such spheroid networks develop a highly three-dimensional morphology with cell clusters up to 60 μm in thickness and are interconnected by pronounced bundles of neuronal fibers and glial processes. We could reliably record from up to hundreds of neurons simultaneously per culture for ≤90 d. By quantifying the formation of these three-dimensional structures over time, while regularly monitoring electrical activity, we were able to establish a strong link between spheroid morphology and network activity. In particular, the formation of cell clusters accelerates formation and maturation of correlated network activity. Astrocytes both influence electrophysiological network activity as well as accelerate the transition from single cell layers to cluster formation. Higher concentrations of astrocytes also have a strong effect of modulating synchronized network activity. This approach thus represents a practical alternative to often complex and heterogeneous organoids, providing easy access to activity within a brain-like 3D environment.