Vitamin D is a molecule, now seen as a hormone, it plays an important role in bone health, it is also associated with many diseases, such as diabetes, cancer and cardiovascular problems. 25- hydroxyvitamin D [25(OH)D] is used to estimate vitamin D status because its long half-life. However, to understand the pathophysiological role of vitamin D metabolites on various diseases, we need to evaluate all relevant metabolome. For this reason, a metabolomic panel is required to evaluate the vitamin D status that measurement of 25 (OH) D and other vitamin D biomarkers, e.g., 1α,25 (OH)2 D3 , 3-epi-25 (OH) D, 24,25 (OH)₂ D₃, vitamin D binding protein (DBP), free/bioavailable 25 (OH) D and parathyroid hormone. Mass spectrometers are promising for comprehensive analysis of vitamin D metabolites in the future.