Non-small cell lung cancer (NSCLC) poses a significant global health burden with unsatisfactory survival rates, despite advancements in diagnostic and therapeutic modalities. Novel therapeutic approaches are urgently required to improve patient outcomes. Pharmacological ascorbate (P-AscH⁻; ascorbate at millimolar concentration in plasma) emerged as a potential candidate for cancer therapy for recent decades. In this present study, we explore the anti-cancer effects of P-AscH⁻ on NSCLC and elucidate its underlying mechanisms. P-AscH⁻ treatment induces formation of cellular oxidative distress; disrupts cellular bioenergetics; and leads to induction of apoptotic cell death and ultimately reduction in clonogenic survival. Remarkably, DNA and DNA damage response machineries are identified as vulnerable targets for P-AscH⁻ in NSCLC therapy. Treatments with P-AscH⁻ increase the formation of DNA damage and replication stress markers while inducing mislocalization of DNA repair machineries. The cytotoxic and genotoxic effects of P-AscH⁻ on NSCLC were reversed by co-treatment with catalase, highlighting the roles of extracellular hydrogen peroxide in anti-cancer activities of P-AscH⁻. The data from this current research advance our understanding of P-AscH⁻ in cancer treatment and support its potential clinical use as a therapeutic option for NSCLC therapy.