HyPer, a ratiometric genetically encoded fluorescent sensor, is a popular tool for intracellular hydrogen peroxide detection. When expressed in cultured cells, the freely diffusing version of the sensor (HyPer-cyto) detects temporal patterns of H2O2 generation. However, rapid diffusion of the probe within the nucleocytoplasmic compartment averages the H 2O2 signal even in cases of local oxidant production. Consequently, we immobilized the sensor within specific subcellular compartments allowing it to monitor local increases in H2O2. Here, we provide a protocol of ratiometric imaging and ImageJ-based quantification of H2O2 microdomains produced by cells upon physiological stimulation.