A novel flow-through solid phase spectroscopic assembly implemented with photochemically induced fluorescence (PIF) has been developed for the rapid, sensitive and selective determination of imidacloprid. The pesticide is derivatized on-line by irradiation with ultraviolet light providing an intensively fluorescent compound. The determination is carried out by measuring the fluorescence intensity of the photoproduct once retained on C 18 silica gel filling the flow-cell. The method proposed has been applied to the determination of imidacloprid in natural waters and peppers. The quantification limit (QL) (0.015 mg kg −1) in peppers was lower than the maximum residue limit (MRL) established by Spanish Legislation (0.5 mg kg −1). The method also seems to be suitable for environmental water analysis providing satisfactory recoveries (94.0–108.6%). The results obtained in the analysis of real samples are in good agreement with those provided by a reference liquid chromatography (HPLC) method.