In vitro models of neuronal networks have proven to be a valuable tool for gaining deeper insights into the intricate mechanisms governing the human brain. Notably, the integration of human induced pluripotent stem cells (hiPSCs) with micro-electrode arrays (MEAs) provides a controlled in vitro environment to replicate and analyse both the structural and functional elements of the human brain. As neuronal communication relies on the emission of electrical (and chemical) stimuli, the employment of electrical stimulation becomes crucial for a comprehensive exploration of neuronal assemblies, to better understand their inherent electrophysiological dynamics. However, the establishment of standardized stimulation protocols for cultures derived from hiPSCs is still lacking, thereby hindering the precise delineation of efficacious parameters to elicit responses. To fill this gap, this study aims to investigate the stimulation parameters aiming to induce independent and reliable evoked responses in human-derived neurons. We utilized biphasic voltage pulses with variable amplitudes (1.5, 2, 2.5, and 3V) delivered at different frequencies (0.1 and 0.2Hz). As an experimental model, we exploited glutamatergic (excitatory, E) and GABAergic neurons (inhibitory, I) to construct fully excitatory (E:I 100:0) and mixed (E:I 75:25) cortical networks derived from hiPSCs coupled to MEAs. This approach allowed us not only to optimize stimulation parameters but also to understand their specific impact on the functional dynamics of both excitatory and inhibitory elements within the neuronal networks. This study represents a stepping-stone in the exploration of efficacious stimulation parameters, thus broadening the electrophysiological activity profiling of neural networks sourced from hiPSCs.