1,25-dihydroxyvitamin D3 is the active metabolite of vitamin D3, and has been found to play important roles in mineral balance and immunological regulation. Recently, 1,25-dihydroxyvitamin D3 and similar synthetic analogs in development have been found to possess potential therapeutic values in the treatment of proliferative diseases, including leukemia, breast cancer, and psoriasis. Current methods for the analysis of these compounds may either lack the necessary sensitivity (i.e., UV detection) or specificity (i.e., receptor binding assay). In addition, the synthetic analogs developed to date have not been found to uniformly bind with receptors in binding assays, a property that may render them to be difficult for measurement at the trace levels. Methods utilizing mass spectrometry (MS) have been used for either structural identification or quantification. Electron impact ionization has been the technique of choice for structural analysis, whereas newer ionization techniques have been utilized for the quantification of metabolites, often in conjunction with on-line separation. In this review, MS and non-MS based methods for vitamin D analysis will be discussed, with an emphasis on the former. Historical perspectives, the present state, and the future role of MS in vitamin D research will be discussed. © 1996 John Wiley & Sons, Inc.