Introduction <p>The gut microbiome is a complex ecosystem stratified that varies along different sections of the gut. It comprises a wide array of metabolites originating from both food, host, and microbes. Microbially-derived metabolites, such as bile acids, short-chain fatty acids, and indole derivatives, are of significant interest due to their direct interactions with host physiology and regulating function. Most current studies on the gut microbiome focus on fecal samples, which do not fully represent the upper parts of the gut due to its stratification. To collect microbiome samples from the proximal gut microbiome, endoscopic methods or new non-invasive medical devices can be used.</p> Objectives <p>To enable comprehensive profiling of the gut metabolome and analyze key metabolites, we developed a combined approach combining untargeted and semi-targeted metabolomics using a Q-Exactive Plus Orbitrap mass spectrometer.</p> Methods <p>Initially, we selected 49 metabolites of interest for the gut metabolome based on four distinct criteria. We validated these metabolites through repeatability and linearity tests and created a compound database using the software TraceFinder (ThermoFisher Scientific). For untargeted metabolomics, we established a workflow for the annotation and discovery of molecules.</p> Results <p>Finally, 37 metabolites were validated for semi-targeted metabolomics, and we conducted a proof of concept on small intestinal and fecal samples form a clinical trial (NCT05477069).</p> Conclusion <p>Our combined approach, facilitated by molecular networking, demonstrated the potential to discover new metabolites.</p>