Abstract: We disclose methods for measuring vitamin D metabolite in plasma or serum samples. The methods comprise a step of adding to the plasma or serum samples a non-competitive displacement agent comprising 8-anilino-1-naphthalenesulfonic acid ammonium salt, 3-(acetonylbenzyl)-4-hydroxycoumarin and a water miscible solvent. The non-competitive displacement agent separates vitamin D metabolite from binding proteins in the sample, such that the displaced vitamin D metabolite is available for capture and detection in subsequent binding assays. Thus, our invention finds use in methods of separating and detecting vitamin D metabolites otherwise tightly bound to plasma or serum binding proteins.