Description:Urinary extracellular vesicles (uEVs) offer a non-invasive source of biomarkers for kidney and urinary tract health, but progress in uEV-based metabolomics is hindered by inconsistent isolation and extraction workflows. This project established an experimentally validated, standardizable workflow for robust untargeted metabolite profiling of human uEVs. By systematically benchmarking commonly used approaches and evaluating their impact on vesicle characteristics and metabolite coverage, we identified a practical combination of conditions that maximizes recovery of biologically relevant small EVs and broadens metabolite detection. The resulting optimised workflow supports more reproducible uEV metabolomics and strengthens the foundation for EV-based biomarker discovery.