ABSTRACT Introduction: The high performance liquid chromatography is a technique used for quantification of amino acids in plasma. The definition of reference intervals in the population is very important for the diagnosis and monitoring of individuals with amino acid disorders. Objectives: The objectives of this study were to validate a method for amino acids quantification and define reference intervals in Brazilian children. Results: Good chromatographic separation was achieved using C18 solid-core column. The method showed satisfactory linearity, limits of detection and quantification, precision and accuracy. The reference ranges for aspartate, glutamate, asparagine, histidine, serine, glutamine, arginine, tyrosine, alanine, tryptophan, methionine, valine, phenylalanine, isoleucine and leucine were defined in a group of 60 healthy individuals aged 2 to 14 years. Conclusion: The presented technique can be applied in clinical practice. Each laboratory should preferably establish its own reference intervals. If that is not possible, it is recommended the use of the reference intervals described in this study for the diagnosis and monitoring of Brazilian children in this age group.