The number of assessments of circulating 25-hydroxyvitamin D [25(OH)D] for diagnostic purposes has increased significantly during the past 5 y. Over the years, techniques for quantifying 25(OH)D have increased and evolved. Some changes have been for the better and some have not. Most current methods appear to provide valid results as long as the operator of the given technique is properly trained and motivated. In addition, any laboratory that assesses circulating 25(OH)D for clinical diagnosis needs to participate in the Vitamin D External Quality Assessment Scheme. Finally, research has shown that circulating 25(OH)D is extremely stable in stored serum or plasma samples; this characteristic makes accurate, long-term epidemiologic studies of circulating 25(OH)D possible. In this article, I provide an overview of the techniques available for measuring 25(OH)D, compare these techniques with one another, and assess their clinical utility. I also briefly discuss the stability of 25(OH)D in biological media and present an overview of the Vitamin D External Quality Assessment Scheme.