The superoxide (O2-)-generating enzyme complex of phagocytes, known as the NADPH oxidase, can be assayed in a number of in vitro cell-free (or broken cell) systems. These consist of a mixture of the individual components of the NADPH oxidase, derived from resting phagocytes or in the form of purified recombinant proteins, exposed to an activating agent (or situation), in the presence of NADPH and oxygen. O2- produced by the mixture is measured by being trapped immediately after its generation with an appropriate acceptor in a kinetic assay, which permits the calculation of the linear rate of O2- production over time. Cell-free assays are distinguished from whole-cell assays or assays performed on membranes derived from stimulated cells by the fact that all components in the reaction are derived from resting, nonstimulated cells and, thus, the steps of NADPH oxidase activation (precatalytic [assembly] and catalytic) occur in vitro. Cell-free assays played a paramount role in the identification of the components of the NADPH oxidase complex, the diagnosis of various forms of chronic granulomatous disease (CGD), and, more recently, the analysis of the domains present on the components of the NADPH oxidase participating in protein-protein interactions leading to the assembly of the active complex.