Metabolomics aims at the identification and quantification of key-end point metabolites, basically polar, in order to study changes in biochemical activities in response to pathophysiological stimuli or genetic modifications. Targeted profiling assays have enjoyed a growing popularity during the last years with LC-MS/MS as a powerful tool for development of such (semi-) quantitative methods for a large number of metabolites. Here we describe a method for absolute quantification of ca. 100 metabolites belonging to key metabolite classes such as sugars, amino acids, nucleotides, organic acids, and amines with a hydrophilic interaction liquid chromatography (HILIC) system comprised of ultra (high) performance liquid chromatography (UHPLC) with detection on a triple-quadrupole mass spectrometer operating in both positive and negative electrospray ionization modes.