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Background: The reactive oxygen species (ROS) generated in the biological systems play an important role in pathological conditions and specific normal cellular processes, such as signaling pathways and drug sensitivity. Methods: Intracellular ROS was determined using (2'-7'dichlorofluorescin diacetate) DCFH-DA fluorimetric probe, malondialdehyde as lipid peroxidation index detected by the Thiobarbituric acid reactive substances method. Cells' protein carbonyl contents were assessed with 2,4-Dinitrophenylhydrazine (DNPH) derivatization spectrophotometrically at 360-385 nm. Results: The oxidative stress induces ROS (p?0.05), MDA (Malondialdehyde) (p?0.05), and protein carbonylation (p?0.01) was significantly higher in HER2-positive BT-474, SK-BR-3, and MDA-MB-453 compared to the HER2-negative MDA-MB-231 and MCF-7 cell lines. Conclusion: We hypothesized that increased oxidative stress in HER2-positive cell lines is due to the oncogenic function of the HER2 and PI3K/Akt signaling activation, resulting in glycolysis induction. It is assumed that HER2-positive cell lines with high ROS levels are more vulnerable to further damage by increased ROS levels induced by pro-oxidant anticancer agents.