Rationale :The emergence of new mass spectrometry (MS) dissociation methods has highlighted lipid isomers as new biomarkers. Only a few commercial methods without derivatization are available to characterize phosphatidylcholines (PCs) at the isomeric level. We propose to use electron transfer dissociation (ETD) as a method to determine the position of both double bonds and stereo numbering ( sn ) on glycerol for PC species. Methods :Doubly charged PCs were analyzed using alkali salts to promote the formation of [PC + 2Alk] 2+ species. ETD‐MS 2 experiments were performed using a quadrupole ion trap mass spectrometer with an ESI source to annotate sn ‐positions. ETD‐CID‐MS 3 experiments were then done on ETnoD [M + 2Alk] +• or [M + 2Alk−N(CH 3 ) 3 ] +• species to localize double bond positions. Density functional theory (DFT) calculations and cyclic ion mobility spectrometry c‐IMS‐MS/MS experiments were used to support fragmentation assumptions. Results : ETD‐MS 2 experiments exhibit a systematic sn ‐2 favorable cleavage, allowing sn ‐positioning through a radical cation‐driven mechanism supported by DFT calculations. This behavior contrasts with CID experiments, in which the initial positioning of alkali cations influences ester bond cleavage, as shown by c‐IMS‐MS/MS experiments. The dissociation process studied for ETD‐CID‐MS 3 experiments on 10 different PC isomers allowed us to localize double bonds for either monounsaturated or polyunsaturated species. Conclusions : The dissociation rules established for ETD‐MS 2 and ETD‐CID‐MS 3 experiments on PC isomers enable their annotation at the isomeric level without instrumental modification or complex sample preparation. This method could be easily coupled to LC separation using post‐column introduction of an alkali salt for complex mixture analysis.