A real time method to measure intracellular H2O2 would be very impactful in characterizing rapid changes that occur in physiologic and pathophysiologic states. Current methods do not provide the sensitivity, specificity and spatiotemporal resolution needed for such experiments on intact cells. We developed the use of HyPer, a genetic indicator for H2O2 that can be expressed in the cytosol (cyto-HyPer) or the mitochondria (mito-HyPer) of live cells. INS-1 cells or islets were permeabilized and the cytosolic HyPer signal was a linear function of extracellular H2O2, allowing fluorescent cyto-HyPer signals to be converted to H2O2 concentrations. Glucose increased cytosolic H2O2, an effect that was suppressed by overexpression of catalase. Large perturbations in pH can influence the HyPer signal, but inclusion of HEPES in the perfusate prevented pH changes, but did not affect glucose-induced cyto-HyPer signals suggesting this effect is largely pH-independent. Using the assay, two fundamental questions were addressed. Knockdown of SOD2, the mitochondrial form of SOD, completely suppressed glucose-induced H2O2. Further, glucose also induced mitochondrial superoxide and H2O2 production, which preceded the appearance of cytosolic H2O2. Therefore, glucose-induced H2O2 largely originated from mitochondria. Finally, glucose-induced HyPer signal was less than 1/20th of that induced by toxic levels of H2O2. Overall, the use of HyPer for real time imaging allowed resolution of acute changes in intracellular levels of H2O2 and will have great utility for islet studies involving mechanisms of H2O2 mediating signaling and oxidative stress.