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HyPer is a genetically encoded fluorogenic sensor for hydrogen peroxide which is generally used for the ratiometric imaging of H2O2 fluxes in living cells. Here, we demonstrate the advantages of HyPer-based ratiometric flow cytometry assay for H2O2, by using K562 and human mesenchymal stem cell lines expressing HyPer. We show that flow cytometry analysis is suitable to detect HyPer response to submicromolar concentrations of extracellularly added H2O2 that is much lower than concentrations addressed previously in the other HyPer-based assays (such as cell imaging or fluorimetry). Suggested technique is also much more sensitive to hydrogen peroxide than the widespread flow cytometry assay exploiting H2O2-reactive dye H2DCFDA and, contrary to the H2DCFDA-based assay, can be employed for the kinetic studies of H2O2 utilization by cells, including measurements of the rate constants of H2O2 removal. In addition, flow cytometry multi-parameter ratiometric measurements enable rapid and high-throughput detection of endogenously generated H2O2 in different subpopulations of HyPer-expressing cells. To sum up, HyPer can be used in multi-parameter flow cytometry studies as a highly sensitive indicator of intracellular H2O2.Ratiometric flow cytometry (FC) analysis is applicable for HyPer-based studies.FC is suitable to detect submicromolar concentrations of extracellularly added H2O2.FC can be employed for the measurements of the rate constants of H2O2 removal.FC is suitable to detect changes in the level of endogenously generated H2O2.Multi-parameter FC analysis enables detection of H2O2 in different cell populations.