25-hydroxyvitamin D, 25(OH)D is the most abundant vitamin D metabolite in the circulation, representing the best indicator of the nutritional status of this fat-soluble vitamin. Two distinct forms exist: 25(OH)D3 from cutaneously derived vitamin D (cholecalciferol), the predominant natural source of vitamin D in humans and 25(OH)D2 from vitamin D2 (ergocalciferol), derived almost entirely from supplementation or fortification of food.1 Worldwide, there has been an explosion of interest in the physiological, pathological, therapeutic and laboratory aspects of 25(OH)D. Request for its measurement has increased dramatically over the last few years with an annual increase of about 80-90%.2,3 At the Clinical Biochemistry Laboratory of the Royal Hospital, Muscat, Sultanate of Oman, the annual request rate for serum 25(OH)D during 2009 was at a much higher degree compared to 2007. The vast majority of patients tested were deficient in 25(OH)D. There is growing awareness for the role of vitamin D; not only for its role in metabolic bone disease, but also, the increasing recognition for its association with a variety of diseases. Several randomized controlled trials have revealed that vitamin D deficiency has been linked to the development of different chronic diseases such as cardiovascular diseases, autoimmune diseases, diabetes mellitus, neuromuscular dysfunction, chronic kidney diseases, different cancers, infections, and gynecological problems.4,5 Data from these studies mentioned earlier have demonstrated that circulating vitamin D is an important reflector of the total mortality risk.6 A recent prospective cohort study by Zittermann et al. in a specialized heart centre revealed that patients in the lowest quintiles of 1,25-dihydroxyvitamin D (1,25(OH)D, also termed calcitriol) and 25(OH)D were more likely to have coronary heart disease, heart failure, hypertension, diabetes mellitus or renal failure compared to patients with higher concentrations of 25(OH)D. The study also showed that low serum concentrations of 1,25(OH)D and 25(OH)D were related to higher 1-year mortality risk, while there was a significant decrease in 1-year mortality risk in patients with higher serum concentrations of vitamin D. The results were also consistent in patients representing different risk factors and multivariate risk adjustments such as age, body mass, smoking, aspirin use, renal function, inflammatory markers, and various co-morbidities.7 Other studies such as the study by Dobnig et al. which focused on the levels of vitamin D and cardiovascular mortality and a study by Wolf et al. which studied the levels of vitamin D and mortality in patients on hemodialysis also showed similar findings and have also confirmed such an association.6,8 Furthermore, meta analysis of different randomized controlled trials have revealed that vitamin D supplementation has been linked to lower total mortality in subjects with low 25(OH)D concentrations compared with un-supplemented individuals.9 Thus, it is worth providing 25(OH)D therapy or supplementation to high risk individuals without necessarily measuring their serum 25(OH)D concentrations, which may not be available at many laboratories. In addition to the increasing awareness regarding the key role of 25(OH)D in the maintenance of many physiological processes and recognition of its deficiency as a growing health problem, an analytical verification has to be addressed. Although there is no consensus on the optimal levels of serum 25(OH)D, most experts recommend that the standard level which confers its optimum physiological protective role and provides the full advantages of vitamin D health benefits is ≥75nmol/L (30 ng/ml).10 Vitamin D status has been defined at different 25(OH)D cut-offs, with levels: 50-74nmol/L (20-30 ng/ml) as suboptimal, 25-49 nmol/L (10-20 ng/ml) as insufficiency, 375nmol/L (150 ng/ml) or even at higher levels.5,10 With the use of such definitions, it has been estimated that one billion people worldwide have vitamin D deficiency or insufficiency, 40-100% of US and European elderly men and women are deficient in vitamin D and more than 50% of postmenopausal women taking medication for osteoporosis had suboptimal 25(OH)D levels