Vitamin D 25-Hydroxy is used to determine if bone weakness, bone malformation, or abnormal metabolism of calcium (reflected by abnormal calcium, phosphorus, PTH) is occurring as a result of a deficiency or excess of vitamin D.

Since vitamin D is a fat-soluble vitamin and is absorbed from the intestine like a fat, vitamin D is sometimes used to monitor individuals with diseases that interfere with fat absorption, such as cystic fibrosis and Crohn's disease, and in patients who have had gastric bypass surgery and may not be able to absorb enough vitamin D. Vitamin D is sometimes used to determine effectiveness of treatment when vitamin D, calcium, phosphorus, and/or magnesium supplementation is prescribed.