In recent years, ambient ionization mass spectrometry (AIMS) including laser ablation rapid evaporation IMS, has enabled direct biofluid metabolome analysis. AIMS procedures are, however, still hampered by both analytical, i.e., matrix effects, and practical, i.e., sample transport stability, drawbacks that impede metabolome coverage. In this study, we aimed at developing biofluid-specific metabolome sampling membranes (MetaSAMPs) that offer a directly applicable and stabilizing substrate for AIMS. Customized rectal, salivary, and urinary MetaSAMPs consisting of electrospun (nano)fibrous membranes of blended hydrophilic (polyvinylpyrrolidone and polyacrylonitrile) and lipophilic (polystyrene) polymers supported metabolite absorption, adsorption, and desorption. Moreover, MetaSAMP demonstrated superior metabolome coverage and transport stability compared to crude biofluid analysis and was successfully validated in two pediatric cohorts (MetaBEAse, n = 234 and OPERA, n = 101). By integrating anthropometric and (patho)physiological with MetaSAMP-AIMS metabolome data, we obtained substantial weight-driven predictions and clinical correlations. In conclusion, MetaSAMP holds great clinical application potential for on-the-spot metabolic health stratification.