It is important to assess pediatric patients for nutritional deficiency when they are receiving specific interventions, such as enteral feeding. We focused on measurement of C0 and 3-hydroxyisovalerylcarnitine (C5-OH) with tandem mass spectrometry (MS/MS), which is performed as part of the newborn mass screening. The purpose of this study was to investigate the usefulness of MS/MS for screening carnitine and biotin deficiencies. Forty-two children (24 boys, 18 girls) were enrolled between December 2013 and December 2015. Blood tests, including measurement of serum free carnitine via the enzyme cycling method, and acylcarnitine analysis on MS/MS of dried blood spot (DBS), were performed for the evaluation of nutrition status. Median patient age was 2 years (range, 2 months-14 years). Mean serum free carnitine was 41.8 ± 19.2 μmol/L. In six of the 42 patients, serum free carnitine was <20 μmol/L (range, 4.0-18.7 μmol/L). C0 and C5-OH measured on MS/MS of DBS were 33.8 ± 20.2 nmol/mL and 0.48 ± 0.22 nmol/mL, respectively. There was a strong positive correlation (r = 0.89, P < 0.001) between serum free carnitine and C0 measured on the same day. In one patient on hydrolyzed formula, C5-OH was >1.00 nmol/L. Therapy-resistant eczema was improved by treatment with additional biotin and a non-hydrolyzed formula. C0 and C5-OH, measured on MS/MS of DBS, were useful for screening carnitine and biotin deficiencies.