In the past quarter century, more than 50 metabolites of vitamin D have been described. To date, only a few of these have been quantified in blood, but this has widened our understanding of the pathologic role that altered vitamin D metabolism plays in the development of diseases of calcium homeostasis. Currently, awareness is growing of the prevalence of vitamin D insufficiency in the general population in association with an increased risk of several diseases. However, for many researchers, it is not clear which vitamin D metabolites should be quantified and what the information gained from such an analysis tells us. Only 2 metabolites, namely, 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D], have received the greatest attention. Of these, the need for measuring serum 1,25(OH)2D is limited, and this metabolite should therefore not be considered as part of the standard vitamin D testing regimen. On the other hand, serum 25(OH)D provides the single best assessment of vitamin D status and thus should be the only vitamin D assay typically performed. Currently, numerous formats exist for measuring serum 25(OH)D concentrations, each with its own advantages and disadvantages. This article reviews the currently available methods for serum 25(OH)D quantitation and considers important issues such as whether both the D2 and the D3 forms of the vitamin should be assayed, whether total or free concentrations are most important, and what measures should be taken to ensure the fidelity of the measurements.