Hydrogen peroxide (H2O2) is not only a key mediator of oxidative stress but also one of the most important cellular second messengers. This small short-lived molecule is involved in the regulation of a wide range of different biological processes, including regulation of cellular signaling pathways. Studying the role of H2O2in living systems would be challenging without modern approaches. A genetically encoded fluorescent biosensor, HyPer, is one of the most effective tools for this purpose. Recent Advances: HyPer has been used by many investigators of redox signaling in various models of different scales: from cytoplasmic subcompartments and single cells to tissues of whole organisms. In many studies, the results obtained using HyPer have enabled a better understanding of the roles of H2O2in these biological processes. However, much remains to be learned. Critical Issues: In this review, we focus on the uses of HyPer. We provide a general description of HyPer and its improved versions. Separate chapters are devoted to the results obtained by various groups who have used this biosensor for their experiments in living cells and organisms. Future Directions: HyPer is an effective tool for H2O2imaging in living systems as indicated by the increasing numbers of publications each year since its development. However, this biosensor requires further improvements. In particular, much brighter and more pH-stable versions of HyPer are necessary for imaging in mammalian tissues.