The present invention relates to a method for releasing bound vitamin D by bringing a sample containing vitamin D into contact with a release reagent which contains at least one hydrotropic substance and at least one transition metal salt. The released vitamin D can subsequently be determined quantitatively. The present invention relates further to a reagent for releasing and optionally determining vitamin D, containing at least one hydrotropic substance and at least one transition metal salt, as well as to the use of such a release reagent for releasing and optionally determining vitamin D.