5-Methyltetrahydrofolate (5MTHF) is the active one-carbon donor and the principal circulating form of plasma folates. It is involved in a number of metabolic and neurodevelopmental processes and analysis of cerebrospinal fluid (CSF) 5MTHF is of great importance in the diagnosis of cerebral folate deficiency (CFD). Serum 5MTHF levels and the 5MTHF serum/CSF ratio may be important additional parameters for the understanding of CFD. We developed a HPLC method for the measurement of 5MTHF in serum and established reference values for the pediatric population. Serum samples from 64 healthy children were extracted with Sep-Pak C18 cartridges and 5MTHF was separated by RP-HPLC and quantified by electrochemical detection. 5MTHF was separated from other folates and detected after 8.7 min with linearity of up to 1600 nmol/L. The detection limit was 4.5 nmol/L and recovery during solid-phase extraction for low and high concentrations of 5MTHF was 66 and 62%, respectively. Within-run imprecision (13.5%) was slightly higher than run-to-run imprecision (8.5%). 5MTHF levels in healthy children were found to be age-dependent, decreasing from 158.0 nmol/L in newborns to 60.1 nmol/L in children older than 16 years. The method we describe is sensitive, selective, and reliable for the analysis of 5MTHF from 400 microL of serum.