A b s t r a c t A r t i c l e I n f oVitamin D was discovered about one century ago, which is part of the family of hormones. Vitamin D supplements have been commonly used in medical prescription. Deficiency of Vitamin D 3 has been related to various severe diseases, including cardiovascular disease, cancer and diabetes. Therefore, fast and reliable analytic methods for Vitamin D are very important for clinical diagnosis and treatment, as well as for biomedical research. To date, immunoassay, LC-MS/MS and HPLC are three major methods for the qualification and quantitation of Vitamin D. In this paper, we will go through the advantage, disadvantage and limits of each method and provide advice for future analysis of this group of hormone.