The inability of current mass spectrometry techniques to differentiate phospholipid isomers results in a routine under-estimation of phospholipid molecular diversity in complex biological matrices. Recent technological advances in tandem mass spectrometry and ion activation are helping to overcome these limitations, but all rely on tandem mass spectrometry with unit mass-selection and suffer from co-isolation of isobaric or isomeric species. Accordingly, separation of phospholipid isomers and isobars prior to characterization is required to fully delve into the complexity of the lipidome. Here we present a novel two-stage workflow combining reversed-phase ultra-high performance liquid chromatography with ozone-induced dissociation (OzID) and combined-collision- and ozone-induced-dissociation (COzID) that reduces spectral complexity and enables discrimination of lipid isomers and isobars. Application of this technique to the analysis of human red blood cell lipid extracts allowed the separation, or partial separation, of adduct ion and head group isobars as well as double bond and sn-positional isomers affording near complete structural characterization of low abundance lipids, e.g. PC 18:0/20:3(n-6), PS 18:0/20:4(n-6) and PS 20:4(n-6)/18:0 all observed at m/z 834.7. We also introduce a software plug-in that automatically annotates OzID mass spectra to assign the carbon–carbon double bond positions in lipids. This new workflow allows us to delve deeper into the lipidome and represents another valuable tool for the lipidomics toolbox.