<p>The antidiabetic drug metformin possesses antioxidant and cell protective effects including in neuronal cells, suggesting its potential use for treating neurodegenerative diseases. This study aimed to assess metformin’s effects on viability and antioxidant activity in human-induced pluripotent stem cell (hiPSC)-derived neurons under varying concentrations and stress conditions. Six lines of hiPSC-derived neuronal progenitors derived from healthy human iPSCs were treated with metformin (1-500 µM) on day 18 of differentiation. For mature neurons (day 30), three concentrations (10 µM, 50 µM, and 100 µM) were used to assess cytotoxicity. MG132 proteasomal inhibitor and sodium arsenite (NaArs) were used to investigate oxidative stress, and 50 µM of metformin was tested for its protective effects against oxidative stress in hiPSC-derived neurons. Metformin treatment did not affect cell viability, neuronal differentiation, or trigger reactive oxygen species (ROS) generation in healthy hiPSC-derived motor neurons. Additionally, mitochondrial membrane potential (MMP) loss was not observed at 50 µM metformin. Metformin effectively protected neurons from stress agents and elevated the expression of antioxidant genes when treated with MG132. However, an interplay between MG132 and metformin resulted in lower expression of Nrf2 and NQO1 compared to the MG132 group alone, indicating reduced JC-1 aggregate levels due to MG132 proteasomal inhibition. Metformin upregulated antioxidant genes in hiPSC-derived neurons under stress conditions and protected the cells from oxidative damage.</p>