Neuronal networks derived from human induced pluripotent stem cells (hiPSCs) have gained attention for their potential use in in vitro modeling of epilepsy and seizure-like electrophysiological activity. Similar to in vivo animal models, seizure-like activity can be induced in these neuronal cultures using kainic acid (KA), direct agonist of the glutamate receptors. However, to establish more relevant in vitro model using KA, better understanding of KA mediated acute and prolonged effects on human neuronal networks is required. In this study, we investigated both acute and long-term KA concentration-responses on expression levels of glutamate receptor genes, cell viability, and electrophysiological activity in human cortical neuronal networks. Microelectrode array (MEA) measurements were utilized to assess neuronal functionality. Furthermore, the recovery and behavior of the networks after washout was assessed. The results reveal distinct concentration-dependent responses on both electrophysiological and gene expression levels. Tested high KA concentrations did not permanently compromise neuronal functionality or viability, as the activity recovered after KA washout. The study depicts detailed KA mediated mechanisms on neuronal functionality and further supports the utilization of KA in developing more physiologically relevant in vitro models of seizures and epilepsy.