In vitro models combined with human induced pluripotent stem cells (hiPSCs) are a widely used tool to investigate the complex mechanism of the human brain. Indeed, the use of hiPSCs-derived neuronal networks allow to create in vitro patient-specific models that most closely resembles the processes of the human system, providing a reliable model to test neuroactive properties of different compounds. This study investigates how different drugs affected the electrophysiological activity of in vitro systems composed by excitatory (E) and inhibitory (I) neurons derived from hiPSCs. Our experimental model included three different culture configurations: 100% excitatory neurons (100E), 75% excitatory and 25% inhibitory neurons (75E25I), and 100% inhibitory neurons (100I). To these neuronal networks, 2-amino-5-phosphonovaleric (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and bicuculline (BIC) were administrated. The recordings of the spontaneous and chemically stimulated electrophysiological activity were performed by exploiting Micro-Electrodes Arrays (MEAs). The administration of APV and CNQX resulted in an unaffected firing activity, while led to a decrease of the bursting and network bursting activity of both 100E and 75E25I configurations. On the other hand, BIC led to a significant increase of the firing activity of the 100I and 75E25I, and of the bursting and network bursting activity of the 75E25I configuration. This comprehensive investigation sheds light on the different effects of APV, CNQX and BIC on the electrophysiological dynamics of neuronal networks with different excitatory-inhibitory compositions. These findings contribute to our understanding of drug-induced modulation in neuronal circuits and provide valuable insights for potential therapeutic investigations in neurological disorders.