Bacteria colonizing the human intestinal tract exhibit a high phylogenetic diversity that reflects their immense metabolic potentials. The catalytic activity of gut microbes has an important impact on gastrointestinal (GI) functions and host health. The microbial conversion of carbohydrates and other food components leads to the formation of a large number of compounds that affect the host metabolome and have beneficial or adverse effects on human health. Metabolomics is a metabolic-biology system approach focused on the metabolic responses understanding of living systems to physio-pathological stimuli by using multivariate statistical data on human body fluids obtained by different instrumental techniques. A metabolomic approach based on an analytical platform could be able to separate, detect, characterize and quantify a wide range of metabolites and its metabolic pathways. This approach has been recently applied to study the metabolic changes triggered in the gut microbiota by specific diet components and diet variations, specific diseases, probiotic and synbiotic food intake. This review describes the metabolomic data obtained by analyzing human fluids by using different techniques and particularly Gas Chromatography Mass Spectrometry Solid-phase Micro Extraction (GC-MS/SPME), Proton Nuclear Magnetic Resonance ((1)H-NMR) Spectroscopy and Fourier Transform Infrared (FTIR) Spectroscopy. This instrumental approach has a good potential in the identification and detection of specific food intake and diseases biomarkers.