Introduction: Diet and dietary interventions have drawn considerable attention in the literature as etiopathogenic factors and therapeutic approaches to Autism Spectrum Disorders (ASD), respectively. The objective of this study was to determine the influence of a gluten-free, casein-free (GFCF) diet on the alterations of behavior in children and adolescents diagnosed with ASD. We also aim to explore the possible association between ASD symptoms and urinary concentrations of beta-casomorphin. Methods: A total of 28 patients were recruited for this crossover clinical trial. Patients followed a normal diet (including gluten and casein) for three months and a GFCF diet for another three months. The order of the intervention--i.e., beginning with normal diet or with GFCF diet--was determined at random. The subjects were evaluated at three time points: at the onset of the study, after the first diet, and after the second diet. Each evaluation entailed three questionnaires on behavior and autism, a food frequency questionnaire (to determine adherence to the diet) and a determination of concentrations of beta-casomorphin in urine using chromatographic techniques. Results: No significant behavioral changes were found after a GFCF diet. No association was found between ASD symptoms and urinary concentrations of beta-casomorphin. Conclusion: A GFCF diet followed for three months do neither show significant changes in behavioral symptoms of autism nor in urine concentrations of beta-casomorphin using chromatographic detection. However, we need further studies including elements of placebo and double-blindness and more sophisticated beta-casomorphin detection techniques to better define subjects who might benefit from these diets.