This chapter provides an overview of versatile and efficient chemical syntheses of vitamin D derivatives by application of either linear or convergent synthesis ap‐-proaches. Synthesis of the most relevant naturally occurring vitamin D metabolites and their deuterated counterparts to use as calibration and reference standards in LC‐-MS/MS assays is also shown. The chapter then summarizes the most important mass spectrometric approaches to quantify important vitamin D metabolites in human biofluids. In addition, new developments are described that are aimed at the pathobio‐-logical interpretation of the measured vitamin D metabolite distributions in various human diseases.