Background: Previous studies have shown that the measurement of vitamin D and its derivatives, especially its active metabolite 1α, 25-dihydroxy-vitamin D [1,25(OH) 2 D], is highly complex and prone to analytical error. We have evaluated a new immunological method for detecting and quantifying of 1,25(OH) 2 D. This assay is fully automated, sensitive and uses a specific recombinant fusion protein for capturing of 1,25(OH) 2 D. The assay was originally developed by DiaSorin for the immunoassay analyzer LIAISON XL. Methods: Performance data of this assay were determined including intra- and inter-assay precision, recovery, linearity, and limit of detection of the DiaSorin 1,25(OH) 2 D immunoassay on the LIAISON XL analyzer. Respective data were compared from two different liquid chromatography tandem mass spectrometry (LC-MS/MS) assays and a common radioimmunoassay (RIA) using clinical samples taken from patients suffering from vitamin D deficiency, chronic renal failure, biliary atresia, hyperparathyroidism, vitamin D-dependent rickets or sarcoidosis, as well as from pregnant women and high-level athletes. Results: The performance evaluation of 1,25(OH) 2 D resulted in an intra-assay and total imprecision correlation variant between 1.4% and 5.2% and 3.8%–7.1% with the new immunoassay and 3.5%–5.8% or 3.8%–7.5% with the LC-MS/MS method, respectively. Limits of detection and quantification of the immunoassay were 0.7 ng/L and 5.0 ng/L for the LIAISON XL immunoassay and 1.8 ng/L and 5.4 ng/L for the LC-MS/MS assay, respectively. Pearson’s coefficients of correlation were 0.998 and 0.952 for method comparison to different established LC-MS/MS methods. Linear regression according to Passing and Bablok showed larger deviations to the RIA (slopes 0.64–0.97, coefficients of correlation 0.822–0.823). Conclusions: The DiaSorin LIAISON XL 1,25(OH) 2 D immunoassay appears to have improved comparability to LC-MS/MS with low imprecision and limits of detection. The assay time of 65 min, the small sample volume required (75 μL) and the throughput of 90 tests/hour without manually handling time for extraction and purification procedures is superior to the LC-MS/MS method.