Purpose: The measurement uncertainty is a quantitative indicator of the quality of the test result obtained and indicates how the test results represent the actual value. For this reason, we aimed to calculate the measurement uncertainty for the 25-hydroxyvitamin D3 assay, which was analyzed by liquid chromatography-tandem mass spectrometry in our laboratory, to compare our findings with other results in the literature and to evaluate the possible effect of uncertainty value on patients' D vitamin status. Materials and Methods: For 25-hydroxyvitamine D3 analyzed by liquid chromatography-tandem mass spectrometry, in the direction of the measurement uncertainty calculation model, which is based on the Nordest guide, standard uncertainties arising from 20 external quality controls, including 4 cycles, 5 levels per cycle, resulting from a total of 440 internal quality controls pathological and normal levels at one year, and the combined uncertainty was calculated by taking this value as a square root. Then the extended uncertainty of measurement was obtained by multiplying with the combined standard uncertainty value k factor. Results: The intra-laboratory reproducibility was 7.44%, the bias from the laboratory was 16.01%, the bias from the measurement procedure was 0.82%, the measurement uncertainty of the bias component was 16.03%, the combined standard uncertainty was 17.67% The uncertainty of measurement was calculated as 34.64%. Conclusions: For 25-hydroxyvitamin D3 the total allowable error is +/- 25%, as a result of our study; The expanded measurement uncertainty value for 25-hydroxyvitamin D3 was found to be high. Targeted solutions should be sought to limit possible errors in order to reduce this value to less than 25%. Adding measurement uncertainty to the patient's outcome will increase confidence in the physician's strength and laboratory results at the point of clinical decision-making.