Certain rare inborn errors of metabolism clinically present with intractable seizures that readily respond to vitamin therapy. If identified early, brain damage due to seizures can be prevented. A LC-MS method was developed and validated for the simultaneous quantification of the biomarkers in selected vitamin responsive pediatric seizures from dried blood spots. Application of the validated method to a seizure cohort of 46 patients indicated strong agreement of the method for clinical validity. Reference intervals for these biomarkers in dried blood spots were also determined for the population, after screening 956 neonates. The developed method was seen to be sensitive, linear, accurate and precise for testing vitamin responsive pediatric seizures.