The superoxide (O2·−)-generating NADPH oxidase complex of phagocytes comprises a membrane-associated heterodimeric flavocytochrome, known as cytochrome b558 (consisting of NOX2 and p22phox) and four cytosolic regulatory proteins, p47phox, p67phox, p40phox, and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2·− generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome, a process known as NADPH oxidase assembly. A consequent conformational change in NOX2 initiates the electron flow along a redox gradient, from NADPH to molecular oxygen (O2), leading to the one-electron reduction of O2 to O2·−. Historically, methodological difficulties in the study of the assembled complex derived from stimulated cells, due to its lack of stability, led to the design of “cell-free” systems (also known as “broken cells” or in vitro systems). In a major paradigm shift, the cell-free systems have as their starting point NADPH oxidase components derived from resting (unstimulated) phagocytes, or as in the predominant method at present, recombinant proteins representing the components of the NADPH oxidase complex. In cell-free systems, membrane receptor stimulation and the signal transduction sequence are absent, the accent being placed on the actual process of assembly, all of which takes place in vitro. Thus, a mixture of the individual components of the NADPH oxidase is exposed in vitro to an activating agent, the most common being anionic amphiphiles, resulting in the formation of a complex between cytochrome b558 and the cytosolic components and O2·− generation in the presence of NADPH. Alternative activating pathways require posttranslational modification of oxidase components or modifying the phospholipid milieu surrounding cytochrome b558. Activation is commonly quantified by measuring the primary product of the reaction, O2·−, trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of rates of O2·− production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount role in the identification and characterization of the components of the NADPH oxidase complex, the performance of structure–function studies, the deciphering of the mechanisms of assembly, the search for inhibitory drugs, and the diagnosis of various forms of chronic granulomatous disease (CGD).