Vitamin D is essential for maintaining a healthy mineral and bone metabolism. Currently, six forms of vitamin D are known. They range from vitamin D2 to vitamin D7 and are distinguished by their differing side chains. Only vitamin D2 and vitamin D3 have been found to be biologically active based on the elucidation of activation pathways. Deficiencies in Vitamin D cause rickets and many other health problems, including reduced muscle function, autoimmune diseases, cardiovascular disease, and some cancers. The following chapter describes a simple and reproducible method for the analysis of Vitamin D via liquid chromatography with triple quadrupole mass spectrometry.