Application of genetically encoded biosensors of redox-active compounds promotes the elaboration of new methods for investigation of intracellular redox activities. Previously, we have developed a method to measure quantitatively the intracellular concentration of hydrogen peroxide (H₂O₂) in living cells using genetically encoded biosensor HyPer. In the present study, we refined the method and applied it for comparing the antioxidant system potency in human cells of different phenotypes by measuring the gradient between the extracellular and cytoplasmic H₂O₂ concentrations under conditions of H₂O₂-induced external oxidative stress. The measurements were performed using cancer cell lines (K-562 and HeLa), as well as normal human cells – all expressing HyPer in the cell cytoplasm. As normal cells, we used three isogenic lines of different phenotypes – mesenchymal stem/stromal cells (MSCs), induced pluripotent stem cells (iPSCs) derived from MSCs by reprogramming, and differentiated iPSC progenies with the phenotype resembling precursory MSCs. When exposing cells to exogenous H₂O₂, we showed that at low oxidative loads (<50 μM of H₂O₂) the gradient depended on extracellular H₂O₂ concentration. At high loads (>50 μM of H₂O₂), which caused the exhaustion of thioredoxin activity in the cell cytoplasm, the gradient stabilized, pointing out that it is the functional status of the thioredoxin-depended enzymatic system that drives the dependence of the H₂O₂ gradient on the oxidative load in human cells. At high H₂O₂ concentrations, the cytoplasmic H₂O₂ level in cancer cells was found to be several hundred times lower than the extracellular one. At the same time, in normal cells, extracellular-to-intracellular gradient amounted to thousands of times. Upon reprogramming, the potency of cellular antioxidant defense increased. In contrast, differentiation of iPSCs did not result in the changes in antioxidant system activity in the cell cytoplasm, assuming that intensification of the H₂O₂-detoxification processes is inherent to a period of early human development.