Ultraviolet photodissociation (UVPD) mass spectrometry was used to characterize the structures of amphiphilic glycosphingolipids and gangliosides in comparison to collision induced dissociation (CID) and higher energy collision dissociation (HCD) in a high performance Orbitrap mass spectrometer. UVPD produced the widest array of fragment ions diagnostic for both the ceramide base and oligosaccharide moieties. CID and HCD generated mainly glycosidic B/Y and C/Z cleavages of the oligosaccharides moieties and very few informative fragments related to the hydrophobic ceramide base. Several unique cleavages at the sphingoid base and the fatty acid chain occurred upon UVPD, as well as a wider variety of cross ring cleavages (A/X ions), thus affording differentiation of isobaric gangliosides. An LC-UVPD-MS strategy allowed the elucidation of 27 gangliosides among five different classes.