There is increasing evidence that diseases caused by dysfunctional mitochondria (MD) are associated with autism spectrum disorder (ASD). A comprehensive meta-analysis showed that developmental regression was reported in half of the children with ASD and mitochondrial dysfunction which is much higher than in the general population of ASD. The aim of the present exploratory study was to determine lactate concentrations in urine of children with ASD, as a non-invasive large-scale screening method for metabolic abnormalities including mitochondrial dysfunction and its possible association with regression. First, clinical characteristics of MD were examined in 99 children (3-11 years) with ASD. Second, clinical characteristics of MD, severity of ASD and reported regression were compared between children with the 20% lowest lactate concentrations and those with the 20% highest lactate concentrations in urine. Third, clinical characteristics of MD and lactate concentration in urine were compared in children with (n = 37) and without (n = 62) reported regression. An association of urine lactate concentrations with mitochondrial dysfunction and regression could not be demonstrated in our large ASD cohort. However, since ASD children were reported by their parents to show a broad range of phenotypic characteristics of MD (e.g., gastro-intestinal and respiratory impairments), and lactate concentrations in urine are not always increased in individuals with MD, the presence of milder mitochondrial dysfunction cannot be excluded. Development of alternative biomarkers and their implementation in prospective studies following developmental trajectories of infants at elevated likelihood for ASD will be needed in the future to further unravel the association of ASD with mitochondrial dysfunction and eventually improve early detection.