Vitamin D testing is part of laboratory practice since more than 30 years but has become a routine parameter only recently, due to a highly increasing amount of research in the field resulting in new clinical applications. Vitamin D actually represents a family of molecules of which 25OH Vitamin D and 1,25(OH)2 Vitamin D, under their D3 and D2 forms, are the most important to date. Physical detection methods and immunoassays exist for both molecules and are being reviewed and discussed. New developments in the measurement of C3-epi-25OH Vitamin D, 24,25(OH)2 Vitamin D, and free/bioavailable 25OH Vitamin D are also presented. The future of Vitamin D testing is considered based on the evolution of laboratories and based on the scientific research that is currently performed.