Developing alternative MS/MS strategies to distinguish isomeric lipids has become a high impact goal in shotgun lipidomics. Novel approaches have been developed to resolve structural features that are not discernible by traditional shotgun methods and have consequently promoted the discovery of new disease biomarkers. However, these methods have largely been limited to characterizing lipids with low structural complexity. Here, ultraviolet photodissociation (UVPD) strategies for phospholipid characterization are expanded for analysis of cardiolipins (CL), a class of phospholipids that exhibits a higher degree of structural complexity. A hybrid collision induced dissociation/193 nm UVPD (CID/UVPD) approach was implemented to pinpoint the location of both double bond and cyclopropyl unsaturations on the four acyl chains of CLs. This strategy was complemented with CID for the de novo elucidation of unknown CLs in biological extracts.