Six laboratories associated with the Vitamin D Standardization Program (VDSP) participated in an interlaboratory comparison of LC with tandem MS (MS/MS) methods for the determination of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] in human serum. The laboratories analyzed two different serum-based Standard Reference Materials (SRMs) intended for use in the determination of 25-hydroxyvitamin D and 30 samples from the Vitamin D External Quality Assessment Scheme (DEQAS). All laboratory methods for 24,25(OH)2D3 were based on isotope dilution LC-MS/MS; three of the methods used derivatization of the vitamin D metabolites before LC-MS/MS. Laboratory results were compared to the National Institute of Standards and Technology (NIST) results, which were obtained using their newly developed candidate reference measurement procedure for 24,25(OH)2D3. Laboratory results for the SRM samples varied in comparability to the NIST results, with one laboratory in excellent agreement (-1.6% mean bias), three laboratories at 10-15% mean bias, and the remaining laboratory at 36% mean bias. For the 30 DEQAS samples, the mean bias for the five laboratories ranged from 6 to 15%; however, the SD of the bias ranged from 8 to 29%. As a result of this intercomparison study, one laboratory discovered and corrected a method calculation error and another laboratory modified and improved their LC-MS/MS method.