1α,25-dihydroxy vitamin D [1,25(OH)(2)D] is the active metabolite of vitamin D. Antibody-based detection methods lack specificity, but when combined with isotope dilution/ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry, immunoextraction provides an attractive method for 1,25(OH)(2)D. We developed a method for simultaneous quantification of 1,25(OH)(2)D(2) and 1,25(OH)(2)D(3) with a 4.6-min instrument cycle time. Results are available 36 h after sample preparation begins. Sample preparation consisted of protein precipitation, immunoextraction with solid-phase anti-1,25(OH)(2)D antibody, and derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione. Analytes were resolved using reversed-phase UPLC and quantified using positive ion electrospray ionization-tandem mass spectrometry. We used hexadeuterated 1,25(OH)(2)D(3) and 1,25(OH)(2)D(2) as internal standards and performed method comparisons against the DiaSorin RIA and an LC-MS/MS method available at a reference laboratory. 1,25(OH)(2)D(3) intraassay and interassay imprecision was 5.6% and 8.0% (120 pmol/L) and 8.7% and 13% (48 pmol/L). Limits of detection and quantification were 1.5 pmol/L and 3.0 pmol/L, respectively. 1,25(OH)(2)D(2) intraassay and interassay imprecision was 8.7% and 11% (186 pmol/L) and 11% and 13% (58 pmol/L). Limits of detection and quantification were both 1.5 pmol/L. Comparison with RIA had a proportional bias of 0.75, constant bias of -4.1, and Pearson correlation (r(2)) of 0.31. Comparison with a reference LC-MS/MS assay had a proportional bias of 0.89, constant bias of 3.7, and r(2) of 0.88. Protein precipitation with antibody-based extraction is effective for sample preparation before LC-MS/MS analysis of derivatized 1,25(OH)(2)D. This method appears to have improved specificity over a clinically used RIA with low imprecision and limits of detection.