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Canavan disease is a serious rare neurometabolic disorder characteristic of an accumulation of N-acetylaspartic acid (NAA) in biological fluids. Its determination mainly in urine is the key part of the diagnostic procedure. Despite the several advantages of using dried blood spot (DBS) samples for clinical analysis, it has not been reported for this purpose so far. Therefore, this work was focused on the development of a HPLC–MS/MS method for the determination of NAA in a dried blood spot. The method using a stable isotope-labeled internal standard was elaborated and partially validated, with an estimated limit of quantification of 0.06 μmol dm−3, and with intra-day and inter-day precisions up to 4.1 and 8.0%, respectively. The elaborated method was applied for the NAA determination in healthy controls and in two siblings with Canavan disease. There were three quantification approaches evaluated using one-point calibration as well as calibration curves with and without matrix. The results correlated very well and exhibited parallelism of one-point calibration and calibration curve approaches. The mean NAA concentrations determined in DBS of control newborns and older children (10 months–7 years) were 0.9 and 0.5 μmol dm−3, respectively. Patients were clearly distinguished from controls, considering the NAA concentrations were more than 10 times higher than in an aged-matched control group.