We describe a new approach applicable to the determination of organic acids that serve as diagnostic markers for several inherited metabolic disorders. We utilized liquid chromatography/tandem mass spectrometry for analysis of organic acid derivatives of a recently described benzofurazan reagent. The derivatization step was necessary to obtain organic acid derivatives suitable for analysis by reversed-phase liquid chromatography with high ionization efficiency for mass spectrometry in the positive-ion mode. In this work, a group of related dicarboxylic acid markers containing five or six carbon atoms were analyzed and validation was performed for glutaric and 3-hydroxyglutaric acids, the specific markers for glutaric acidemia type 1. Derivatization was achieved by reacting untreated urine with the derivatization reagent under mild conditions. The reaction mixture was analyzed on a C18 ultra-performance liquid chromatography (UPLC) column (50x2.1 mm, 1.7 microm) and detected in the multiple reaction monitoring mode in 5 min. Calibration curves were linear up to at least 1000 microM with detection limits for glutaric and 3-hydroxyglutaric acids of 0.025 and 0.02 microM, respectively (signal-to-noise ratio of 3). Intra-day (n=11) and inter-day (n=6) coefficients of variation were better than 11.2%. The assay was successfully applied to control (n=134) and glutaric acidemia type 1 (n=55) urine samples.