BACKGROUND 25-hydroxyvitamin D [25(OH)D] assays are characterized by poor between-assay comparability. This result emphasizes the need for reference measurement procedures (RMPs) to establish calibration traceability and assist in method validation. We aimed at developing candidate RMPs on the basis of isotope- dilution liquid chromatography–tandem mass spectrometry (ID-LC-MS/MS) for separate quantification of serum 25(OH)D2 and 25(OH)D3. METHODS Hexa-deuterated 25(OH)D3/D2 was added to serum. This mixture was extracted with n-hexane and fractionated on Sephadex LH-20 before 2-dimensional LC-MS/MS. In the first dimension, both procedures used a C4 column; however, in the second dimension, the 25(OH)D2 procedure used a C18 and the 25(OH)D3 procedure used a Zorbax SB-CN column. Calibration was traceable to the NIST Standard Reference Material (SRM) 2972. Validation comprised assessment of interference and limit of quantification/detection. Imprecision and trueness were validated by analysis of the SRM 972 against specifications (CV <5% and bias <1.7%). The expanded uncertainty for quadruplicate measurements was estimated. RESULTS Testing of potentially interfering substances was negative. Interference by 3-epi-25(OH)D3 was resolved by sufficient chromatographic resolution. The limits of quantification/detection were 1.1 nmol/L and 0.09 pmol/L for 25(OH)D3 and 1.2 nmol/L and 0.05 pmol/L for 25(OH)D2. Mean total CVs and differences from the SRM 972 target (± 1-sided 95% CI) were 2.1% and 1.1% ± 1.5% [25(OH)D3] and 3% and 1.3% ± 0.6% [25(OH)D2], respectively. The respective expanded uncertainties were 3.4% and 3.9%. CONCLUSIONS From the validation data, we conclude that we achieved our objective of 2 state-of-the-art candidate RMPs for serum 25(OH)D3 and 25(OH)D2.