The present invention relates to a method of measuring a vitamin D metabolite in a sample, the method comprising the steps of (a) treating said sample with a vitamin D metabolite releasing reagent under conditions appropriate to release a vitamin D metabolite from vitamin D-binding protein and not to cause protein precipitation, (b) subjecting the treated sample obtained in step (a) to a chromatographic separation, and (c) measuring a vitamin D metabolite during or after said chromatographic separation. The present invention also relates to methods for determining the vitamin D status of a subject, for use in the diagnosis of disease, and to agents and kits for use in performing the methods of the invention.