Untargeted metabolomics provides a comprehensive platform for identifying metabolites whose levels are altered between two or more populations. By using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS), hundreds to thousands of peaks with a unique m/z ratio and retention time are routinely detected from most biological samples in an untargeted profiling experiment. Each peak, termed a metabolomic feature, can be characterized on the basis of its accurate mass, retention time and tandem mass spectral fragmentation pattern. Here a seven-step protocol is suggested for such a characterization by using the METLIN metabolite database. The protocol starts from untargeted metabolomic LC-Q-TOF-MS data that have been analyzed with the bioinformatics program XCMS, and it describes a strategy for selecting interesting features as well as performing subsequent targeted tandem MS. The seven steps described will require 2–4 h to complete per feature, depending on the compound.