Fecal metabolomics-based analysis indisputably constitutes a very useful tool for elucidating the biochemistry of digestion and absorption of the gastrointestinal system. Fecal samples represent the most suitable, non-invasive, specimen for the study of the symbiotic relationship between the host and the intestinal microbiota.It is well established that the balance of the intestinal microbiota changes in response to some stimuli, physiological such as gender, age, diet, exercise and pathological such as gastrointestinal and hepatic disease. Fecal samples have been analyzed using the most widespread analytical techniques, namely, NMR spectroscopy, GC-MS, and LC-MS/MS. Rat fecal sample is a frequently used and particularly useful substrate for metabolomics-based studies in related fields. The complexity and diversity of the nature of fecal samples require careful and skillful handling for the effective quantitative extraction of the metabolites while avoiding their deterioration. Parameters such as the fecal sample weight to extraction solvent volume, the nature and the pH value of the extraction solvent, and the homogenization process are some important factors for the optimal extraction of samples, in order to obtain high-quality metabolic fingerprints, using either untargeted or targeted metabolomics.