The fluorescent sensor HyPer allows monitoring of intracellular H2O2levels with a high degree of sensitivity and specificity. Here, we provide a detailed protocol of ratiometric imaging of H2O2produced by cells during phagocytosis, including instructions for experiments on different commercial confocal systems, namely, Leica SP2, Leica SP5, and Carl Zeiss LSM, as well as wide-field Leica 6000 microscope. The general experimental scheme is easily adaptable for imaging H2O2production by various cell types under a variety of conditions. © 2013 Elsevier Inc. All rights reserved.