Vitamin D is a group of steroids that have various effects on the human body and support the immune system. This application shows LC/MS/MS separation of the hydroxylated and non-hydroxylated forms of vitamin D2 and vitamin D3. The amount of their hydroxylated forms determines the total vitamin D in a blood sample. Non-hydroxylated forms of vitamin D2 and vitamin D3 are important for food analysis. The LC/MS/MS chromatogram shows the separation of all the above mentioned analytes.