We report a novel approach to derivatize the primary, secondary, and tertiary hydroxy group(s) of oxysterols with N,N-dimethylglycine (DMG) in the presence of both 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide and 4-(N,N-dimethylamino)pyridine to yield their corresponding mono- or di-DMG esters. Eight oxysterols including 7-oxocholesterol, 5α,6α- and 5β,6β-epoxycholesterols, as well as 7α-, 7β-, 24(S)-, 25-, and 27-hydroxycholesterols, were studied. Electrospray ionization tandem mass spectrometric characterization of these singly or doubly protonated derivatives demonstrates the presence of an informative fragmentation pattern for each oxysterol derivative. Potential dissociation pathways for the production of these unique fragmentation patterns are proposed and discussed. Collectively, these informative and unique fragmentation patterns allow rapid and direct discrimination of the identities of 7α-, 7β-, 24(S)-, 25-, and 27-hydroxycholesterol isomers, as well as 5α,6α- and 5β,6β-epoxycholesterol isomers, thereby potentially providing a foundation for quantitative analysis of oxysterols in biological samples in combination with a chromatographic separation.