The analysis of 25-hydroxyvitamin D3 (25(OH)D3) and related metabolites represents a considerable challenge for both clinical and research laboratories worldwide. There is currently debate about the best methodology employed to assess vitamin D status and whether the 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3) should be separated and quantitated when measuring 25(OH)D3. Mass spectrometry techniques are generally regarded as the gold standard due to high specificity for vitamin D metabolites. However, many methods require high sample volumes for analysis. We have developed a new 2 dimensional (2D) ultra performance liquid chromatography (UPLC) separation coupled tandem mass spectrometry (MS/MS) detection to accurately quantitate 25(OH)D3, epi-25(OH)D3, and 25(OH)D2 in adults and children, requiring only 50 μL of human serum. The assay gives excellent separation of epi-25(OH)D3, and 25(OH)D2 from 25(OH)D3, has excellent precision with an intra-assay CV of 0.5 % at 74 nmol/L and can report down to 2 nmol/L for 25(OH)D3. Furthermore, the method shows excellent agreement with the vitamin D external quality assessment scheme (DEQAS) quality control program for vitamin D analysis. We present this approach as a candidate reference method for 25(OH)D3, epi-25(OH)D3, and 25(OH)D2 analysis in humans.