Background: Biomarkers that reflect glioblastoma tumour activity and treatment response are urgently needed to help guide clinical management, particularly for recurrent disease. As the urinary system is a major clearance route of circulating extracellular vesicles (EVs; 30-1000 nm nanoparticles) we explored whether sampling urinary-EVs could serve as a simple and non-invasive liquid biopsy approach for measuring glioblastoma-associated biomarkers. Methods: Fifty urine specimens (15-60 ml) were collected from 24 catheterised glioblastoma patients immediately prior to primary (n=17) and recurrence (n=7) surgeries, following gross total resection (n=9), and from age/gender-matched healthy participants (n=14). EVs isolated by differential ultracentrifugation were characterised and extracted proteomes were analysed by high-resolution data-independent acquisition liquid chromatography tandem mass spectrometry (DIA-LC-MS/MS). Results: Overall, 6857 proteins were confidently identified in urinary-EVs (q-value≤0.01), including 94 EV marker proteins. Glioblastoma-specific proteomic signatures were determined, and putative urinary-EV biomarkers corresponding to tumour burden and recurrence were identified (FC≥|2|, adjust p-val≤0.05, AUC>0.9). Conclusion: In-depth DIA-LC-MS/MS characterisation of urinary-EVs substantiates urine as a viable source of glioblastoma biomarkers. The promising ‘liquid gold’ biomarker panels described here warrant further investigation.