The disclosure relates to methods for measuring levels of 25(OH)vitamin D (250HD) in a mammalian fluid sample. The disclosure further relates to kits for measuring levels of 25(OH)vitamin D (250HD) in a mammalian fluid, the procedure comprising the step of release of the vitamin D from the serum binding protein (DBP), capturing of the released analyte by a specific antibody and competitive assay of the captured vitamin D against a conjugate between the vitamin D and a tracer. The observed signal is inversely proportional to vitamin D concentration.