A flow injection analysis (FIA) to quantify imidacloprid was developed based upon (1) ultraviolet (254 nm) photochemical dissociation of imidacloprid to produce nitrite, (2) chemical reduction of the nitrite to nitric oxide by iodide in acid, (3) removal of gas-phase nitric oxide from the aqueous stream using a membrane separator, and (4) detection of the nitric oxide by chemiluminescent reaction with ozone. The cross-reactivity of imidacloprid with eight metabolites of imidacloprid was determined using a commercial ELISA kit and the FIA method. While the ELISA kit demonstrated varying degrees of cross-reactivity, cross-reactivity in the FIA method was observed for only the N-nitro and N-nitroso metabolites. The optimized analytical FIA method, FIA provides a linear response in imidacloprid concentration over four orders of magnitude, has a limit of detection of 5.6 pmol (1.5 ng) of imidacloprid, and exhibits an inter-day precision of 0.4%. Spike-recoveries by FIA demonstrated excellent recovery of imidacloprid in natural waters, hemlock xylem fluid, honey, and grapes with little to no interference from the matrix.