Depression’s link to serotonin dysregulation is well-known. The monoamine theory posits that depression results from impaired serotonin activity, leading to the development of antidepressants targeting serotonin levels. However, their limited efficacy suggests a more complex cause. Recent studies highlight mitochondria as key players in depression’s pathophysiology. Mounting evidence indicates that mitochondrial dysfunction significantly correlates with major depressive disorder (MDD), underscoring its pivotal role in depression. Exploring the serotonin-mitochondrial connection, our study investigated the effects of chronic serotonin treatment on induced-pluripotent stem cell-derived astrocytes and neurons from healthy controls and two case study patients. One was a patient with antidepressant non-responding MDD (“Non-R”) and another had a non-genetic mitochondrial disorder (“Mito”). The results revealed that serotonin altered the expression of genes related to mitochondrial function and dynamics in neurons and had an equalizing effect on calcium homeostasis in astrocytes, while ATP levels seemed increased. Serotonin significantly decreased cytosolic and mitochondrial calcium in neurons. Electrophysiological measurements evidenced that serotonin depolarized the resting membrane potential, increased both sodium and potassium current density and ultimately improved the overall excitability of neurons. Specifically, neurons from the Non-R patient appeared responsive to serotonin in vitro, which seemed to improve neurotransmission. While it is unclear how this translates to the systemic level and AD resistance mechanisms are not fully elucidated, our observations show that despite his treatment resistance, this patient’s cortical neurons are responsive to serotonergic signals. In the Mito patient, evidence suggested that serotonin, by increasing excitability, exacerbated an existing hyperexcitability highlighting the importance of considering mitochondrial disorders in patients with MDD, and avoiding serotonin-increasing medication. Taken together, our findings suggested that serotonin positively affects calcium homeostasis in astrocytes and increases neuronal excitability. The latter effect must be considered carefully, as it could have beneficial or detrimental implications based on individual pathologies.