Nontargeted metabolomics aims to capture as many metabolites as possible present in a biological system under a certain condition. Despite several analytical techniques commonly applied for nontargeted metabolomics, liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS) is one of the most employed techniques. To cover broad metabolic classes, different chromatographic separation techniques are implemented, i.e., reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) techniques. Each method offers a complementary polarity range. While RP covers semi- to nonpolar metabolites, such as fatty acids, acylcarnitines, steroids, bile acids, etc., HILIC allows the analysis of polar metabolites, such as amino acids, amines, and organic acids. Nontargeted metabolomics platforms at Helmholtz Munich have been validated for various biological matrices, including biological liquids (plasma, serum, urine), tissues (liver, heart), and cell samples of human and rodent. Analysis is based on the Sciex ZenoTOF 7600 and three chromatographic separation methods, which can be used individually or in combination.