Natural products (NPs) play a key role in chemical biology and drug development thanks to their high diversity in chemical space. This diversity however renders their separation and universal detection challenging. Recently, ultra-high pressure liquid chromatography (UHPLC) systems have been recognised as the most versatile technique for the efficient separation of NPs in crude complex mixtures. This work firstly investigated the separation of small molecules and peptides in complex natural mixtures by UHPLC and ion mobility spectrometry (IMS). Secondly, a dereplication (rapid identification of known compounds) method was developed for the identification of NPs present in natural extracts by high resolution mass spectrometry (HR-MS) with the combined use of heuristic filters, chemotaxonomic information and a new and original retention prediction method. This thesis showed that the new developments in UHPLC and HR‐MS applied to high resolution metabolite profiling of complex natural matrices give promising perspectives in dereplication and metabolomics.