This project aims to establish novel methods and protocols for unambiguous identification of unknown metabolites by NMR methods and high resolution LC-MSn, starting with plant derived semi-polar compounds. A reproducible MSn spectral tree method to systematically fragment metabolites down to their smallest fragments has been developed. This fragmentation method, using a NanoMate-Orbitrap FT-MS, was used to fragment a series of flavonoid molecules. Differentiation between different isomers was achieved, including isobaric flavononoid-glycosides. The reproducibility of the spectral tree method for detection and putative identification of components in complex biological mixtures was subsequently tested on crude tomato and Arabidopsis extracts using LC-Orbitrap with MSn on-the-fly. Based on similarities of parts of their fragmentation patterns with reference compounds, a large number of conjugated flavonoids present in these samples could be annotated. In addition, a method for automated LC-MS-SPE-NMR of unknown semi-polar compounds in both plants and human fluids has been developed. By using this method on tomato fruit peel extracts, flavonoid molecules have been identified that were not as yet reported in tomatoes.