Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). Here, NOX1 is linked to experimental autoimmune encephalomyelitis.