The prevalence of vitamin D deficiency in the general population has become a major public health problem. Vitamin D deficiency might have significant consequences not only to bone health but possibly to autoimmune-, infectious and cardiovascular disease. This has resulted in increased clinical testing for 25-hydroxyvitamin D (25(OH)D) in serum, as circulating 25(OH)D is regarded as the best indicator of adequate exposure to sunlight and dietary intake of vitamin D. There are reportedly over 50 vitamin D metabolites of which 25(OH)D and 1,25(OH)2D are well known to provide clinical information. More recently, there is increasing interest in measuring the C3-epimer of 25(OH)D, which has shown to contribute significantly to the 25(OH)D concentration, particularly in infant populations, and in 24,25(OH)2D, a major catabolite of 25(OH)D metabolism. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an analytical tool that allows the specific determination of all relevant vitamin D metabolites, with the potential of performing multiple analyte analysis in a single experimental setting, creating a vitamin D profile. This article reviews recent advances in the quantification of vitamin D metabolites using LC-MS/MS.