Superoxide radical is a very important parameter of oxidative stress involved in a variety of biological phenomena; therefore, its in vivo study is of utmost significance. However, its accurate detection is a challenge due to its short lifetime and its very low physiological concentration. All current assays are qualitative and nonspecific, and at best they are performed in vitro. The current dihydroethidine-based assay overcomes all these problems and introduces the following novelties. First, it measures the in vivo superoxide production in animals, plants, and microorganisms. Second, it is ultrasensitive and very simple in that it can measure superoxide radical as low as 1.5 pmol in biological samples as low as 5 mg. Third, the very high sensitivity of the assay renders possible, for the first time, the measurement of the actual rate of formation of superoxide radical under physiological and simulated nonphysiological conditions.