Liquid Chromatography-Mass Spectrometry (LC-MS) untargeted experiments require complex bioinformatic strategies to extract information from the experimental data. Here we discuss the “data preprocessing,” the set of procedures performed on the raw data to produce a data matrix which will be the starting point for the subsequent statistical analysis. Data preprocessing is a crucial step on the path to knowledge extraction, which should be carefully controlled and optimized in order to maximize the output of any untargeted metabolomics investigation.