We provide a clinical and analytical evaluation of the reformulated version of the Abbott Architect 25-hydroxyvitamin D assay. We compared this assay with three commercial automated immunoassays and against a VDSP-traceable liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in six different populations. We also supplemented 40 healthy volunteers with either 600,000 IU of vitamin D2 or 100,000 of vitamin D3 to evaluate the performance of the immunoassays vs. the LC-MS/MS.