Objective: Measurement of circulating 25-hydroxyvitamin D 3 is accepted as the clinical indicator of vitamin D status. It is very important of valid measurement of 25-hydroxyvitamin D 3 as clinical. Therefore there is need a standardization for measurement. Methods except tandem mass spectrometry (LC-MS/MS) measure total vitamin D and are less sensitive. LC-MS/MS is gold standard method for measuring vitamin D values. On this account we have invented a new method and evaluated this method to measure the imprecision and the accuracy and compared it with other systems.