Reactive oxygen species (ROS) are recognized as important signaling components in various processes in plants. ROS are produced for NADPH oxidase in different subcellular compartments and they are involved for a wide range of stimuli, such as cell cycle, growth, plant defenses, abiotic stress responses, and abscisic acid signaling in guard cells. In Arabidopsis, root hairs ROS also play a key role in root hair growth and they control the activity of calcium channels required for polar growth (Takeda et al. Science 319:1241-1244, 2008). The production of reactive oxygen species is under a specific molecular control in order to avoid detrimental side effects. Here we describe a protocol to detect ROS by oxidation of a derivative of fluorescein: 2',7-dihidro dicloro fluorescein (H2DCFDA).