Abstract Background Although correctly used in expanded newborn screening programs to identify patients with possible diseases, flow-injection tandem mass spectrometry (MS/MS) acylcarnitine “profiles” are inadequate for standard clinical uses owing to their limited quantitative accuracy and lack of selectivity. We report the application of our selective, accurate, and precise method for quantification of acylcarnitines, applied to urine glutarylcarnitine from a patient with glutaric acidemia type I (GAI). Methods A previously validated acylcarnitine ultra-HPLC-MS/MS method was used, with a focus on analysis of glutarylcarnitine. Calibrants and samples were isolated by solid-phase extraction and derivatized with pentafluorophenacyl trifluoromethanesulfonate. Acylcarnitine pentafluorophenacyl esters were eluted in 14-min chromatograms. Standardized calibrants and a 13-point, 200-fold concentration range calibration curve were used for accurate quantification of glutarylcarnitine. Quality control samples validated method accuracy and long-term analytic stability. Results Quantification of glutarylcarnitine in urine from a patient with GAI is reported. Long-term analytical stability of the method over a 5-year period is shown. Conclusions Our method for acylcarnitine quantification is shown to be selective, accurate, and precise; thus, we recommend it for confirmatory testing and monitoring of plasma and urine samples from patients with GAI.