Significance: Hydrogen peroxide (H2O2) is a key signaling molecule involved in the regulation of both physiological and pathological cellular processes. Genetically encoded HyPer probes are currently among the most effective approaches for monitoring H2O2dynamics in various biological systems because they can be easily targeted to specific cells and organelles. Since its development in 2006, HyPer has proved to be a robust and powerful tool in redox biology research. Recent Advances: HyPer probes were used in a variety of models to study the role of H2O2in various redox processes. HyPer has been increasingly used in the past few years for in vivo studies, which has already led to many important discoveries, for example, that H2O2plays a key role in the regulation of signaling cascades involved in development and aging, inflammation, regeneration, photosynthetic signaling, and other biological processes. Critical Issues: In this review, we focus on the main achievements in the field of redox biology that have been obtained from in vivo experiments using HyPer probes. Future Directions: Further in vivo studies of the role of H2O2largely depend on the development of more suitable versions of HyPer for in vivo models: Those having brighter fluorescence and a more stable signal in response to physiological changes in pH.