A reversed-phase HPLC method was developed for analyzing sinigrin and gluconasturtiin in six vegetable and two Chinese herb samples. A gradient program and mobile phases using methanol and 0.05% trifluoroacetic acid containing 20 mM ammonium acetate allowed sufficient retention and separation of the glucosinolates in the sample extracts. Quadrupole time-of-flight tandem mass spectrometry in negative ion electrospray ionization was used to analyze the fractions collected from the HPLC elution to confirm the identification of the glucosinolates. The levels of sinigrin and gluconasturtiin in the vegetables and Chinese herbs were determined by using an external calibration method. Concentrations of gluconasturtiin in the Chinese herbs were more than 15 times higher than those of sinigrin. Detection limits were 18 nmol g(-1) for sinigrin and 4 nmol g(-1) for gluconasturtiin when 50 g of fresh vegetable was analyzed.