The metabolome is the complete set of small molecules coming from protein activity (anabolism and catabolism) in living systems. They have a broad range of chemical structures and physico-chemical properties and therefore different analytical methodologies are necessary. Highly polar metabolites, such as sugars and most amino acids are not retained by conventional reversed phase liquid (RP-LC) chromatography columns. Without sufficient retention, coelution may result in identification problems while the detection of compounds by mass spectrometry at low concentrations may also be problematic due to ion suppression. In order to retain compounds based on their hydrophilicity, polar stationary phases and hydrophilic interaction liquid chromatography (HILIC) provide a complementary tool to RP-LC for untargeted comprehensive metabolite fingerprinting. However, robustness of the methods is still limiting their applications. This review focuses on sample pre-treatment, stationary phases, analytical methods and applications for polar compound analysis in biological matrices.