NADPH oxidases (NOX) are superoxide anion radical (O(2)(-•))-generating enzymes. They form a family of seven members, each with a specific tissue distribution. They function as electron transport chains across membranes, using NADPH as electron donor to reduce molecular oxygen to O(2)(-•). NOX have multiple biological functions, ranging from host defense to inflammation and cellular signaling. Measuring NOX activity is crucial in understanding the roles of these enzymes in physiology and pathology. Many of the methods used to measure NOX activity are based on the detection of small molecules that react with NOX-generated O(2)(-•) or its direct dismutation product hydrogen peroxide (H(2)O(2)) to form fluorescent, luminescent, or colored products. Initial techniques were developed to measure the activity of the phagocyte isoform NOX2 during the oxidative burst of stimulated polymorphonuclear leukocytes, which generate large quantities of O(2)(-•). However, other members of the NOX family generate much less O(2)(-•) and hence H(2)O(2), and their activity is difficult to distinguish from other sources of these reactive species. In addition, O(2)(-•) and H(2)O(2) are reactive molecules and most probes are prone to artifacts and therefore should be used with appropriate controls and the data carefully interpreted. This review gives an overview of current methods used to measure NOX activity and NOX-derived O(2)(-•) and H(2)O(2) in cells, tissues, isolated systems, and living organisms, describing the advantages and caveats of many established methods with emphasis on more recent technologies and future perspectives.