While dissociated cultures of rodent neurons on microelectrode arrays (MEAs) have become a prominent method for studying neuronal network properties, establishing human neuronal cultures with mature electrophysiological features compatible with MEAs has posed a significant challenge. The aim of this study was to establish a reliable protocol for generating human-derived neuronal networks on High Density Microelectrode Arrays (HD-MEA) and to characterize their electrophysiological features for potential applications in disease modeling and therapeutic testing. Human-induced pluripotent stem cells (hiPSC) lines were generated from two healthy individuals, as well as a commercial hiPSC line from a control individual. Neuroprogenitor cells (NPCs) were then generated and characterized, from which mixed neuronal cultures were established. Proteomic analysis revealed markers related to neural progenitors, neurons, glia and dynamic shifts in protein expression during differentiation, particularly in pathways associated with neurogenesis, synaptic organization and GABA-shift. Electrophysiological analysis demonstrated an evolution from random activity to bursting behavior, with the application of theta burst stimulation inducing long-term potentiation. Chemical stimulation with bicuculline revealed distinct network dynamics among the lines, suggesting variations in cell composition and excitatory-inhibitory balance. Our findings underscore the potential of hiPSC-derived neuronal networks on HD-MEA for studying synaptic plasticity and neurodevelopmental disorders (NDD). Furthermore, we addressed the need for multiple hiPSC-derived neuronal lines or isogenic sets to accurately identify disease phenotypes, highlighting the importance of considering genetic background in functional analyses. Our study provides a robust experimental platform combining proteomics and electrophysiological analyses, facilitating the investigation of NDD pathophysiology and the development of targeted therapeutics.