Reactive oxygen species (ROS) regulate both normal cell functions by activating a number of enzymatic cascades and pathological processes in many diseases by inducing oxidative stress. For many years since the discovery of ROS in biological systems, there were no adequate methods of detection and quantification of these molecules inside the living cells. We developed the first genetically encoded fluorescent indicator for the intracellular detection of hydrogen peroxide, HyPer, that can be used for imaging of H₂O₂ production by cells under various physiological and pathological conditions. Unlike most known ROS indicators, HyPer allows the generation of a real-time image series that give precise information about the time course and intensity of H₂O₂ changes in any compartment of interest. In this chapter, we describe the method of confocal imaging of hydrogen peroxide production in HeLa cells upon stimulation with epidermal growth factor. The technique described may be accepted with minimal variations for the use in other cell lines upon various conditions leading to H₂O₂ production.