Vitamin D exists in two major forms, vitamin D(3) and vitamin D(2). Vitamin D helps the body absorb calcium and promote optimal bone health. Both forms of vitamin D are metabolized to 25-hydroxyvitamin D in the body, and the levels of 25-hydroxyvitamin D(3) [25(OH)D(3)] and 25-hydroxyvitamin D(2) [25(OH)D(2)] in serum are considered the best indicators of vitamin D status. A candidate reference measurement procedure for serum 25(OH)D(3) and 25(OH)D(2) has been developed and critically evaluated. The deuterated compounds 25(OH)D(3)-d(3) and 25(OH)D(2)-d(3) are used as internal standards for 25(OH)D(3) and 25(OH)D(2), respectively. The 25(OH)D(3) and 25(OH)D(2) and their respective labeled internal standards are simultaneously extracted from serum using liquid-liquid extraction prior to reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chromatographic separation was performed using a cyano (CN) column for both 25(OH)D(3) and 25(OH)D(2). Atmospheric pressure chemical ionization (APCI) in the positive ion mode and multiple reaction monitoring (MRM) were used for LC-MS/MS. The accuracy of the method was evaluated by recovery studies of measuring 25-hydroxyvitamin D [25(OH)D] in spiked samples with known 25(OH)D levels. The recoveries of the added 25(OH)D(3) and 25(OH)D(2) ranged from 99.0% to 101.0%. The absolute recoveries with this method were 97% and 92% for 25(OH)D(3) and 25(OH)D(2), respectively. Excellent precision was obtained with between-set coefficients of variation (CVs) of 0.2-0.6% for 25(OH)D levels >1 ng/g and within 2% for the level of <1 ng/g. Chromatographic separation of 25(OH)D(3) and 25(OH)D(2) from their respective isomers 3-epi-25(OH)D(3) and 3-epi-25(OH)D(2) was achieved. The limit of detection at a signal-to-noise ratio of approximately 3 was 40 pg of 25(OH)D on column (or approximately 0.15 ng/g as expressed as a concentration). This candidate reference measurement procedure for serum 25(OH)D(3) and 25(OH)D(2) demonstrates good accuracy and precision and low susceptibility to interferences. It can be used to provide an accuracy base to which clinical methods for 25(OH)D(3) and 25(OH)D(2) can be compared and that will serve as a standard of higher order for measurement traceability.