This study reports the experimental findings and plasma delivery approach developed at the Plasma Bioscience Research Center, Korea for the assessment of antitumor activity of dielectric barrier discharge (DBD) for cancer treatment. Detailed investigation of biological effects occurring after atmospheric pressure non-thermal (APNT) plasma application during in vitro experiments revealed the role of reactive oxygen species (ROS) in modulation of the antioxidant defense system, cellular metabolic activity, and apoptosis induction in cancer cells. To understand basic cellular mechanisms, we investigated the effects of APNT DBD plasma on antioxidant defense against oxidative stress in various malignant cells as well as normal cells. T98G glioblastoma, SNU80 thyroid carcinoma, KB oral carcinoma and a non-malignant HEK293 embryonic human cell lines were treated with APNT DBD plasma and cellular effects due to reactive oxygen species were observed. Plasma significantly decreased the metabolic viability and clonogenicity of T98G, SNU80, KB and HEK293 cell lines. Enhanced ROS in the cells led to death via alteration of total antioxidant activity, and NADP+/NADPH and GSH/GSSG ratios 24 hours (h) post plasma treatment. This effect was confirmed by annexin V-FITC and propidium iodide staining. These consequences suggested that the failure of antioxidant defense machinery, with compromised redox status, might have led to sensitization of the malignant cells. These findings suggest a promising approach for solid tumor therapy by delivering a lethal dose of APNT plasma to tumor cells while sparing normal healthy tissues.