Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder characterized by the degeneration of motor neurons and presents a critical need for effective treatment. By establishing and characterizing a human-induced pluripotent stem cell (iPSC)-derived motor neuron (iMN) model with dysfunctional TDP-43 nuclear localization signal (ΔNLS), this study investigates the impact of TAR DNA-binding protein 43 (TDP-43) nuclear depletion in ALS. The model successfully recapitulates functional deficits observed in ALS. Proteomic and metabolomic profiling revealed dysregulation in oxidative stress pathways and altered neurotransmitter release, shedding light on the underlying mechanisms of ALS pathophysiology. This work provides a comprehensive understanding of TDP-43 nuclear depletion in the context of ALS pathogenesis.