The plasma/serum concentration of 25-hydroxyvitamin D3 [25(OH)D3] is a diagnostic index for vitamin D deficiency/insufficiency, which is associated with a wide range of diseases, such as rickets, cancer and diabetes. We have reported that the derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) works well in the liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI–MS/MS) assay of the serum/plasma 25(OH)D3 for enhancing the sensitivity and the separation from a potent interfering metabolite, 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3]. However, enhancing the analysis throughput remains an issue in the LC/ESI–MS/MS assay of 25(OH)D3. The most obvious restriction of the LC/MS/MS throughput is the chromatographic run time. In this study, we developed an enhanced throughput method for the determination of the plasma 25(OH)D3 by LC/ESI–MS/MS combined with the derivatization using the triplex (2H0-, 2H3- and 2H6-) DAPTAD isotopologues. After separate derivatization with 1 of 3 different isotopologues, the 3 samples were combined and injected together into LC/ESI–MS/MS. Based on the mass differences between the isotopologues, the derivatized 25(OH)D3 in the 3 different samples were quantified within a single run. The developed method tripled the hourly analysis throughput without sacrificing assay performance, i.e., ease of pretreatment of plasma sample (only deproteinization), limit of quantification (1.0 ng/mL when a 5 μL-plasma was used), precision (intra-assay RSD ≤ 5.9% and inter-assay RSD ≤ 5.5%), accuracy (98.7–102.2%), matrix effects, and capability of separating from an interfering metabolite, 3-epi-25(OH)D3. The multiplexing of samples by the isotopologue derivatization was applied to the analysis of plasma samples of healthy subjects and the developed method was proven to have a satisfactory applicability.