Lipidomics, the systematic study of the lipid composition of a cell or tissue, is an invaluable complement to knowledge gained by genomics and proteomics research. Mass spectrometry provides a means to detect hundreds of lipids in parallel, and this includes low abundance species of lipids. Nevertheless, frequently occurring isobaric and isomeric lipid species complicate lipidomics analyses from an analytical and bioinformatics perspective. Various MS/MS strategies have evolved to resolve ambiguous identifications of lipid species, and these strategies have been supported by corresponding bioinformatics analysis tools. This review intends to familiarize readers with available bioinformatics MS/MS analysis tools and databases, the structural information obtainable from these, and their applicability to different MS/MS strategies. Finally, future challenges in detecting double bond positions are investigated from a bioinformatics perspective.