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Vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes in later life. A method for the quantification of 25-hydroxyvitamin D-3 [25(OH)D-3, the best-established indicator of vitamin D status] in neonatal dried blood spots (DBSs) using LC/ESI-MS/MS has been developed and validated. The method employed two steps of derivatization, a Diels-Alder reaction with 4-phenyl-1,2,4-triazoline- 3,5-dione followed by acetylation, to enhance the detectability of 25(OH)D-3 in ESIMS/MS and to separate 25(OH)D-3 from 3-epi-25-hydroxyvitamin D-3 [3-epi-25(OH)D-3], a potent interfering metabolite. 25(OH)D-3 was extracted from two DBS punches (3mm in diameter, equivalent to 5.3 mu L of whole blood), purified using an Oasis HLB (R) cartridge, and subjected to derivatization prior to analysis with LC/ESI-MS/MS. 25-Hydroxyvitamin D-4 was used as the internal standard. This method was reproducible (intra-and inter-assay RSDs, <6.9%) and accurate (analytical recovery, 95.2-102.7%), and the LOQ was 3.0 ng/mL. The developed method enabled specific quantification of 25(OH)D-3 in neonatal DBSs and detection of vitamin D deficiency without interference from 3-epi-25(OH)D-3.