Metabolomics aims at identification and quantitation 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 enjoying a growing popularity over 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 quantitation 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 with ultra (high) performance liquid chromatography (UHPLC) with detection on a triple quadrupole mass spectrometer operating in both positive and negative modes.