Vitamin D are taken from sunlight exposure and foods, such as oil rich fish. Serum 25-hydroxyvitamin D concentration is most appropriate marker to assess nutritional vitamin D status. On the other hand, 1,25-dihydroxyvitamin D is a clinical marker of disorders in calcium metabolism. To detect vitamin D deficiency and insufficiency, high-throughput and high-sensitive automated measurement system of 25OHD has been developed, and added in health insurance listing. Several methods of 25OHD concentration measurement have been developed based on biochemical or physicochemical such as LC-MS/MS.