The circulating 25(OH)D concentration is the most reliable measure of overall vitamin D status, even though it is biologically inactive. 25(OH)D is measured by commercially available immunoassays, preferably by automated high throughput immunoassay analyzers. However, variability between these methods exists due to different antibodies sources, preliminary extraction or purification procedures, and/or incubation conditions. It is important that these immunoassays utilize antibodies that can react with both 25(OH)D₃ and 25(OH)D₂ equally for accurate assessment of vitamin D status. Alternatively, vitamin D is measured by direct detection methodologies that include both high-pressure liquid chromatography and LC-mass spectrometry (LC-MS). Both offer the advantage of separating and detecting both 25(OH)D₂ and D₃. LC-MS has recently been revitalized as a viable method to measure vitamin D levels. When properly performed, it provides accurate results; however, the throughput, although better than high-pressure liquid chromatography, cannot match the automated immunoassay platforms.

Vitamin D levels are inversely associated with PTH levels and directly associated with intestinal calcium absorption. Therefore, the optimal level of vitamin D is defined as 30 ng/mL, because at this level the PTH begins to level off and intestinal calcium absorption is maximal. Vitamin D levels between 15 and 29 ng/mL are considered insufficient and levels less than 15 ng/mL are considered deficient. Based on these definitions of optimal levels, vitamin D deficiency or insufficiency is highly prevalent worldwide. In contrast, vitamin D intoxication is rare and can occur by inadvertent ingestion of very high doses (>50,000 U), raising serum vitamin D levels to more than 150 ng/mL. It has been shown that doses up to 10,000 U/day for many months do not cause toxicity.

Although 25(OH)D levels reflect the overall vitamin D status, 1-25(OH)₂D levels, even though it is the active metabolite, do not necessarily correlate with vitamin D status. Serum levels of 1-25(OH)₂D do not increase in response to increased intake or exposure to sunlight and are often normal or even elevated in vitamin D insufficiency. Production of 1-25(OH)₂D is tightly regulated by 1α-hydroxylase in the kidneys and the serum level closely correlates with renal function. In end-stage renal disease, this hydroxylation step is severely reduced or negligible. Therefore, circulating concentrations of 1,25(OH)₂D are considerably lower in these patients than in healthy individuals. Inadequately low 1,25(OH)₂D levels decrease serum calcium concentrations and stimulate the production of PTH, resulting in secondary hyperparathyroidism and substantial bone loss. Therefore, the primary utility of measurement of 1,25(OH)₂D is in assessing secondary hyperparathyroidism and hypocalcemia in patients with renal dysfunction. It is also useful in evaluation of patients with hypoparathyroidism, sarcoidosis, and rickets.

Measurement of 1,25(OH)₂D is challenging because of its low circulating levels (normal range 18 to 86 pg/mL) and presence of interfering substances and cross-reactivity with other 1α-hydroxylated vitamin D metabolites. Therefore, chemical extraction (C18 column) and purification of serum samples before assay is necessary. After extraction/purification, 1,25(OH)₂D is measured by radioreceptor or radioimmunoassay. An alternative method is LC-MS, which has been also difficult primarily owing to low levels, lack of ionizable polar groups, and its lipophilic nature. More recently using immunoextraction and concentrating the eluates before LC-MS have been shown to provide a sensitive alternative LC-MS method. Although 25(OH)D measurement serves as the indicator of nutritional vitamin D status, the 1-25(OH)₂D measurement is only indicated in assessing secondary hyperparathyroidism in patients with renal insufficiency.