Urinary organic acids, plasma amino acids and acylcarnitine profile analyses are the main tools used to diagnose inborn errors of metabolisms (IEM).However, without metabolic decompensation, these parameters are often not helpful.On the other hand, in cases of IEM, acylglycines are consistently raised even when patients appear to be in remission.This study aims to set-up a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the determination of urine acylglycines, complementary to organic acid and acylcarnitine profiles, for the diagnosis of IEM.In addition, local reference intervals for various acylglycines are established by using this method.Acylglycines were isolated by solid-phase extraction, derivatized with n-butanol, separated by HPLC, and detected by ESI-MS/MS.Acylglycines were quantified with deuterated internal standards.Mean recoveries of acylglycines ranged from 90.2 to 109.3%.Within-and between-run imprecisions for all acylglycines have CVs less than 10%.Linear regression coefficients were greater than 0.99.Reference intervals were established according to CLSI guidelines by analyzing 204 samples from apparently healthy individuals less than 18 years of age.Partitioning into age group reference intervals was not indicated, according to the Harris and Boyd approach.In this context, a single reference interval for each acylglycine could be used.This method of urine acylglycines analysis is a powerful diagnostic tool, complementary to urine organic acids and plasma acylcarnitine profiling, for detecting certain inborn errors of metabolism.