As clinicians are more widely appreciating the endemic nature of low vitamin D status, measurement of serum 25-hydroxyvitamin D (25(OH)D), the accepted measure of vitamin D status, has increased. Challenges to 25(OH)D measurement include the presence of 2 forms of vitamin D-ergocalciferol and cholecalciferol (vitamin D(2) and vitamin D(3), respectively)- and the hydrophobic nature of vitamin D. The current state of 25(OH)D measurement is reviewed; modest differences between methodologies persist and confound the application of a single cut point (e.g., 30 ng/mL/75 nmol/L) for the diagnosis of low vitamin D status. The absence of standard calibrators contributes to between-laboratory differences in 25(OH)D measurement. Until there is improved assay standardization and subsequent evidence-based consensus, it seems premature to recommend widespread screening 25(OH)D measurement. Selectively obtaining 25(OH)D measurement in individuals at clinical risk for vitamin D deficiency and/or those most likely to promptly experience benefits from supplementation seems appropriate.