Objective: Autism spectrum disorder is defined by repetitive and restricted behaviors and lack of social interactions and commu- nication. It is described as a neurodevelopmental disorder that appears early in life. Autism spectrum disorder was later noted as multifactorial disorder caused by environment, genetic, and other related factors. Amino acids are the precursors of neu- rotransmitters, and they are involved in the pathogenesis of sev- eral diseases and have many functions and effects in the body. They are indicators of protein metabolism and nutritional status. Because of its important properties and its potential relation- ship with autism, this study aimed to investigate serum amino acid levels in 20 healthy children and 25 children with autism spectrum disorder. Methods: Serum amino acid levels were analyzed by liquid chro- matography-mass spectrometry/mass spectrometry. Results: Autism spectrum disorder group had a lower serum concentration of gamma-aminobutyric acid, aspartic acid, and beta-aminoisobutyric acid levels compared to the control group, while glutamic acid, tryptophan, alanine, glycine, proline, ornithine, histidine, and serotonin levels had statistically higher concentrations (P < .05). And also, serum levels of asparagine, arginine, methionine, serine, valine, tyrosine, threonine, lysine leucine, beta alanine, anserine, hydroxyproline, argininosucci- nate, isoleucine, sarcosine, citrulline, histamine, phenylalanine, and glutamine were not found to be statistically lower or higher (P > .05). Conclusion: The study indicated that when compared to the control group, children with autism spectrum disorder had sta- tistically significantly decreased and increased variation in some serum levels of amino acid.