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Background Age, race, and analytic method influence levels of blood amino acids, of which reference intervals are required for the diagnosis and management of inherited metabolic disorders. Objectives To establish age-specific reference intervals for blood amino acids in Thai pediatric population measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods A cross-sectional study of 277 healthy children from birth to 12 years was conducted. Anthropometric, clinical, and dietary information were recorded. Dried blood spots on a filtered paper were used for measurement by derivatized LC-MS/MS. Factors that might affect amino acids such as fasting time and dietary intake were analyzed using quantile regression analysis. Results Levels of thirteen blood amino acids were reported as median and interval from 2.5th–97.5th percentiles. Compared with those of Caucasian, most blood amino acid levels of Thai children were higher. Compared with a previous study using HPLC in Thai children, many amino acid levels are different. Glycine, alanine, leucine/isoleucine, and glutamic acid sharply decreased after birth. Citrulline, arginine, and methionine stayed low from birth throughout childhood, whereas phenylalanine was at middle level and slightly increased during preadolescence. Conclusion Reference intervals of age-specific blood amino acids using LC-MS/MS were established in the Thai pediatric population. They diverge from previous studies, substantiating the recommendation that, for the optimal clinical practice, age-specific reference intervals of amino acids should be designated for the particular population and analysis method.