|Dehydration and inefficient ionization of 1α, 25-DHVD3 and its isomers/analogs in electrospray ionization mass spectrometers (ESI/MS), make them difficult to quantify by this method in a reliable manner. Apart from stability and ionizability a high resolution HPLC method is necessary to separate the isobaric isomers of 1α, 25-DHVD3. To address these challenges a novel reagent has been developed to derivatize 1α,25-DHVD3 and its analogs using a simple and short one step reaction. Detection limit for 1α, 25-DHVD3 in human serum was <5 pg/mL. Isobaric isomers of 1α, 25-DHVD3 were well resolved under chromatographic conditions addressing the interference issue.