The gut microbiome is of tremendous relevance to human health and disease, so it is a hot topic of omics-driven biomedical research. However, a valid identification of gut microbiota-associated molecules in human blood or urine is difficult to achieve. We hypothesize that bowel evacuation is an easy-to-use approach to reveal such metabolites. A non-targeted and modifying group-assisted metabolomics approach (covering 40 types of modifications) was applied to investigate urine samples collected in two independent experiments at various time points before and after laxative use. Fasting over the same time period served as the control condition. As a result, depletion of the fecal microbiome significantly affected the levels of 331 metabolite ions in urine, including 100 modified metabolites. Dominating modifications were glucuronidations, carboxylations, sulfations, adenine conjugations, butyrylations, malonylations, and acetylations. A total of 32 compounds, including common, but also unexpected fecal microbiota-associated metabolites, were annotated. The applied strategy has potential to generate a microbiome-associated metabolite map (M3) of urine from healthy humans, and presumably also other body fluids. Comparative analyses of M3 vs. disease-related metabolite profiles, or therapy-dependent changes may open promising perspectives for human gut microbiome research and diagnostics beyond analyzing feces.