The constantly increasing requests for the measurement of serum 25-hydroxyvitamin D over the last years has led reagent manufacturers to market different automated and semi-automated methods, that being unfortunately not fully harmonized, yield different results. Liquid chromatography coupled to tandem mass spectrometry (LC/MS) has more recently been introduced. This approach allows the distinction between the two forms of 25-hydroxyvitamin D and to measure other metabolites. This approach also requires harmonization to curtail the differences between the different analytical methods. To meet this requirement, the American National Institutes of Health (NIH), the Centre for Disease Control and Prevention (CDC) in Atlanta, the National Institute of Standards and Technology (NIST) and the vitamin D Reference laboratory of Ghent University have pooled their expertise to develop a standardization program. This article reviews the main elements and the difficulties of the automated and semi-automated methods for 25-hydroxyvitamin D, from sample preparation to the analytical phase, as well as those related to mass spectrometry. It also emphasizes the need for standardization to better define the clinical decision thresholds of vitamin D nutritional status.