Vitamin D compounds belong to a group of secosteroids, which occur naturally as vitamin D3 in mammals and D2 in plants. Vitamin D is vital for bone health but recent studies have shown a much wider role in the pathologies of diseases such as diabetes, cancer, autoimmune, neurodegenerative, mental and cardiovascular diseases. Photosynthesis of vitamin D in the human skin and subsequent hepatic and renal metabolism generate a wide range of transformation products occurring over a large dynamic range spanning from picomolar to nanomolar levels. This necessitates selective and sensitive analytical methods to quantitatively capture these low concentration levels in relevant tissues such as blood. Ideally, vitamin D assessment would be performed using a universal and standardized analytical method available to clinical laboratories that provides reliable and accurate quantitative results for all relevant vitamin D metabolites with sufficiently high throughput. At present, LC-MS/MS assays are the most promising techniques for vitamin D analysis. The present review focuses on developments in mass spectrometry methodologies of the past 12 years. It will highlight detrimental influences of the biological matrix, epimer contributions, pitfalls of specific mass spectrometry data acquisition routines (in particular multiple reaction monitoring, MRM), influence of ionization source, derivatization reactions, inter-laboratory comparisons on precision, accuracy, and application range of vitamin D metabolites. © 2013 Wiley Periodicals, Inc. Mass Spec Rev 34: 2–23, 2015.