Subtle structural features in bacterial lipids such as unsaturation elements can have vast biological implications. Cyclopropane rings have been correlated with tolerance to a number of adverse conditions in bacterial phospholipids. They have also been shown to play a major role in Mycobacterium tuberculosis ( M. tuberculosis or Mtb) pathogenesis as they occur in mycolic acids (MAs) in the mycobacterial cell. Traditional collisional activation methods allow elucidation of basic structural features of lipids but fail to reveal the presence and position of cyclopropane rings. Here, we employ 213 nm ultraviolet photodissociation mass spectrometry (UVPD-MS) for structural characterization of cyclopropane rings in bacterial phospholipids and MAs. Upon UVPD, dual cross-ring C-C cleavages on both sides of the cyclopropane ring are observed for cyclopropyl lipids, resulting in diagnostic pairs of fragment ions spaced 14 Da apart, thus enabling cyclopropane localization. These diagnostic pairs of ions corresponding to dual cross-ring cleavage are observed in both negative and positive ion modes and afford localization of multiple cyclopropane rings within a single lipid. This method was integrated with liquid chromatography (LC) for LC/UVPD-MS analysis of cyclopropyl glycerophospholipids in Escherichia coli ( E. coli) and for analysis of MAs in Mycobacterium bovis ( M. bovis) and M. tuberculosis lipid extracts.