Abstract: A method and kit for detecting at least two vitamin D metabolites in a biological sample is disclosed, which comprises processing the biological sample to prepare the sample for LC-MS/MS analysis, passing the prepared sample through a liquid chromatography column having an outlet connected to an inlet port of a tandem mass spectrometer to separate said two vitamin D metabolites, if present in the sample, and introduce the two vitamin D metabolites into the tandem mass spectrometer. The method further comprises generating [M+H]+ ions of each of the two vitamin D metabolites in said tandem mass spectrometer, and generating two fragment ions of said [M+H]+ ions associated with said vitamin D metabolites, wherein said fragment ions are not due to water losses from the [M+H]+ ions; and detecting the fragment ions to identify presence of the two metabolites in the biological sample.