Reactive oxygen species (ROS) play important roles in the initiation and progression of many disease processes. Most fluorescent probes for ROS detection, such as 2,7-dichlorofluorescein (DCFH), function by an oxidative mechanism and are useful for total oxidant detection but are not selective for hydrogen peroxide specifically. Pentafluorobenzenesulfonyl fluorescein is a fluorescent probe that functions by a nonoxidative mechanism. It fluoresces upon perhydrolysis of the sulfonyl linkage, and is selective for hydrogen peroxide over hydroxyl radical, tert-butyl hydroperoxide, superoxide anion, singlet oxygen, and nitrates. Pentafluorobenzenesulfonyl fluorescein can be used as a probe for hydrogen peroxide in whole cell systems.