This study addresses challenges in cerebrospinal fluid (CSF) amino acid profiling in pediatric neurological disorders by establishing age-continuous reference intervals. It examines amino acid variations in epilepsy of different etiologies and evaluates the effects of antiseizure medications (ASMs), resolving inconsistencies in previous research. We retrospectively analyzed 410 CSF samples from pediatric patients (201 with epilepsy). Reference intervals were established using linear regression, adjusting for age and sex as confounders. Statistical analysis of age-normalized data included tests to assess the relationship between clinical features and altered or normal amino acid levels, multiple regression models evaluating the effects of ASMs on CSF amino acid levels, and hierarchical clustering for pattern identification. Age significantly influenced CSF amino acid levels, with higher concentrations observed in neonates, except for aspartic acid and arginine. Sex was not a significant predictor. Over 90 % of patients with epilepsy had normal levels for most amino acids, except for glutamine, which was more frequently elevated in epilepsy. Valproate and GABAA receptor agonists were linked to elevated glutamine, while vigabatrin therapy was associated with increased levels of ornithine, leucine, and isoleucine. Hierarchical clustering identified a subcluster with elevated essential amino acid levels, predominantly comprising patients with generalized seizures or status epilepticus. This study establishes new CSF amino acid reference intervals for pediatric patients and finds no significant associations between individual amino acid levels and epilepsy-related clinical features. However, a subgroup with high essential amino acid levels, primarily associated with generalized epilepsy, suggests potential links to generalized epileptogenic discharges and blood-brain barrier disruption.