In the field of metabolomics, researchers seek to acquire almost complete information about themetabolic composition of a sample to provide fundamental information about the organism’s cel-lular state. In metabolomics analysis today, typically reversed-phase liquid chromatography (LC)is coupled with specific, sensitive, and robust mass spectrometry (MS). That approach, however,misses many moderately polar, and all very polar, compounds; this situation is a problem in plantmetabolomics, because plant metabolites are mainly water-soluble species and thus very polar.Here, we describe new developments in polarity-extended separations using the serial coupling ofreversed-phase LC and hydrophilic interaction liquid chromatography (HILIC) separation steps, in combination with electrospray ionization-time-of-flight-mass spectrometry (ESI-TOF-MS), and theapplication of this approach to plant metabolomics. The resulting retention time vs. mass plots aremolecular fingerprints, as well as sources of further molecular descriptors. Extraction methods,molecular analysis, and data evaluation have to be adapted to the matrix under consideration.Representative strategies using this polarity-extending approach, following so-called suspects andnontargeted screening approaches, are presented