Background: 1α,25-Dihydroxyvitamin D [1,25(OH) 2 vitD] is the bioactive form of vitamin D. Due to the very low concentrations of 1,25(OH) 2 vitD in the blood and its lipophilic character, measurement of this parameter is analytically challenging. Requiring preceding manual extraction steps before analysis, previous assays have been laborious. Methods: In the presented study, we evaluated the performance of two immunoassays from DiaSorin and from Immunodiagnostic Systems (IDS) which combine fully automated extraction and measurement of 1,25(OH) 2 vitD. Imprecision and linearity were verified according to Clinical and Laboratory Standards Institute EP15-A3 and EP6-A guidelines, respectively. Ninety-three patient serum samples sent to our institute for determination of 1,25(OH) 2 vitD, as well as 20 Vitamin D External Quality Assessment Scheme (DEQAS) samples, were used to evaluate correlation and agreement of 1,25(OH) 2 vitD measurements between the two immunoassays and with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Results: Total imprecision was 5.2% or less for the DiaSorin test but reached 20.1% for the IDS iSYS test. 1,25(OH) 2 vitD concentrations measured with the DiaSorin assay showed a strong correlation with 1,25(OH) 2 vitD levels measured by LC-MS/MS and a good agreement with method specific means of DEQAS samples. By contrast, the IDS iSYS test overestimated 1,25(OH) 2 vitD concentrations in human serum, particularly at higher concentrations. Conclusions: Due to its high sensitivity, low imprecision, broad measurement range, and good agreement with 1,25(OH) 2 vitD concentrations measured by LC-MS/MS, the DiaSorin test is a valuable analytical option for the determination of 1,25(OH) 2 vitD.