Autism spectrum disorder (ASD) has been associated with disruptions in tryptophan (TRP) metabolism, affecting the production of key neuroactive metabolites. Investigating these metabolic pathways could yield valuable biomarkers for ASD severity and progression. We included 44 children with ASD and 44 healthy children, members of the same family. The average age in the ASD group was 10.7 years, while the average age in the control group was 9.4 years. Urinary tryptophan metabolites were quantified via liquid chromatography-mass spectrometry operating multiple reaction monitoring (MRM). Urinary creatinine was analyzed on an Advia 2400 analyzer using the Jaffe reaction. Statistical comparisons were made between ASD subgroups based on CARS scores. Our findings indicate that children with ASD have higher TRP concentrations (19.94 vs. 16.91