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.