Spectral library searching is currently the most common approach for compound identification in untargeted metabolomics, with the earliest historical spectral libraries that can be traced back to the 1950s . Metabolite annotation using spectral library searching is based on the concept that molecules undergo fragmentation that creates a reproducible "fingerprint." Matching against a spectral library of ground truth MS/MS spectra collected with chemical standards of known molecules can then be used to narrow down structural hypotheses. During library searching, experimental MS/MS spectra are annotated by matching against the library MS/MS spectra and transferring compound labels from library to experimental spectra when a high-scoring match is achieved. This is the gold standard for metabolite annotation from MS/MS data only, and it forms a level 2 or level 3 annotation based on the guidelines of the Metabolomics Standards Initiative . A level 2 annotation corresponds to library searching resulting in a structural hypothesis for a specific molecule, while a level 3 annotation is a match hypothesis to a molecular family. Especially isomeric compounds with identical precursor mass may result in more than one structural match. For example, it is impossible to distinguish between various stereoisomers of hexenoylcarnitine by MS/MS matching only (Figure ). To promote such a level 3 match to a level 1 identification, complementary analytical approaches, such as nuclear magnetic resonance (NMR), are needed, or all possible isomers in the molecular family have to be tested under the same mass spectrometry conditions to best determine MS/MS spectrum similarity, in addition to liquid chromatography (LC) co-migration of the compound of interest with the chemical standards to validate whether it elutes with the same peak shape and retention time.