The last decade has seen a substantial increase in clinical interest in vitamin D deficiency and of laboratory testing for vitamin D status. Many clinical laboratories in the United States have seen requests for vitamin D testing increase 100% or more in the last 5 years. The most common laboratory test to assess vitamin D nutritional status is total 25-hydroxyvitamin D serum concentrations. Laboratory professionals are often confronted with challenges related to vitamin D testing, including controversy over optimal and target vitamin D concentrations, variable reference ranges across marketed assays and reference laboratories, lack of standardization of vitamin D assays, and misordering of 1,25-dihydroxyvitamin D testing. This article presents a common clinical case scenario regarding vitamin D and an up-to-date discussion and review of the literature on vitamin D testing.