The 25-OH Vitamin D2 and 25-OH Vitamin D3 measurement method was validated using a Zivak Multitasker Fully Automated Sample Preparation and Injection System coupled to a MS detector. The current method incorporates all sample preparations, SPE clean up procedures, LC and MS facilities into an all in one system. This system can report patient data within 6 minutes including the SPE and Sample preparation and mass spectrometry analysis time. The correlation coefficient of the developed method was greater than 0.99 for measurands, 25-OH-vitamin Ds, in the calibration range. The measurement uncertainty of the measurements were evaluated and found as 7.7 % and 7.6 % for 25-OH vitamin D2 and 25-OH-vitamin D3, respectively.