In current practice, patients vitamin D status is assessed by measuring 25(OH)D and interpreting the results against fixed cut-offs of either 50 or 75 nmol/L. However, widely used immunoassay are known for the variable analytical performance and clinical studies indicate that a substantial number of individuals with 25(OH)D serum levels below the cut-offs for deficiency have actually no metabolic or clinical signs of vitamin D deficiency. With the advent of liquid chromatography-tandem mass spectrometry (LC-MS/MS), the accurate quantitation of 25(OH)D and other vitamin D metabolites, such as 24,25(OH)₂D with gold standard technology becomes increasing accessible to clinical laboratories. The possibility of measuring additional vitamin D metabolites allows the assessment of vitamin D status on a functional level. In this presentation it will be shown how the simultaneous measurement of 25(OH)D and 24,25(OH)2D can improve diagnostics in specific patients groups where the established cut-offs are not applicable and where immunoassays fail to deliver accurate results. Finally, a novel approach to diagnose functional vitamin D deficiency will be presented.