The range of applications performed on dried blood spots (DBS) widely broadened during the past decades to now include next-generation sequencing (NGS). Previous publications provided a general overview of NGS capacities on DBS-extracted DNA but did not focus on the identification of specific disorders. We thus aimed to demonstrate that NGS was reliable for detecting pathogenic mutations on genomic material extracted from DBS. Assuming the future implementation of NGS technologies into newborn screening (NBS), we conducted a pilot study on fifteen patients with inherited metabolic disorders. Blood was collected from DBS. Whole-exome sequencing was performed, and sequences were analyzed with a specific focus on genes related to NBS. Results were compared to the known pathogenic mutations previously identified by Sanger sequencing. Causal mutations were readily characterized, and multiple polymorphisms have been identified. According to variant database prediction, an unexplained homozygote pathogenic mutation, unrelated to patient's disorder, was also found in one sample. While amount and quality of DBS-extracted DNA are adequate to identify causal mutations by NGS, bioinformatics analysis revealed critical drawbacks: coverage fluctuations between regions, difficulties in identifying insertions/deletions, and inconsistent reliability of database-referenced variants. Nevertheless, results of this study lead us to consider future perspectives regarding "next-generation" NBS.