To measure urine indolylacroylglycine (IAG) excretion using the IAG:creatinine ratio in children with autism spectrum disorder (ASD) compared with two groups of age matched controls, one with special needs but without ASD (SEN) and one typically developing (TD) and in subgroups with/without current gastrointestinal problems and ASD with and without regression. IAG:creatinine ratio was measured in the urine of 279 children aged 10-14 years: 129 children with ASD (28 with and 101 without regression), 62 SEN controls and 88 TD controls. The prevalence of gastro-intestinal symptoms (GIS) was recorded. No differences were found in the urine IAG:creatinine ratio among groups ASD, TD and SEN; nor in the ASD groups with/without regression, nor in those with/without GIS. This study finds no evidence of increased urine IAG excretion in children with ASD, with or without GIS or with or without regression. Urinary IAG measurements in children with ASD offer no support for increased presence of neuroactive peptides proposed to result from increased gut permeability. We found measurement of urinary IAG to have no value in the diagnosis of autism or in the dietary management of children with ASD.