Most cells release membrane vesicles for various purposes including, but not limited to, intercellular communication and disposal of membrane and soluble proteins. These vesicles are secreted into urine coming from the cells lining the urinary tract and bladder epithelium. These vesicles are a promising source of biomarkers for various cardiovascular and renal diseases. This thesis pursues twofold objectives, one being the development and improvement of an isolation method for urinary membrane vesicles and the second being proteomic characterization of the content of these vesicles. These objectives are important to realise the clinical potential of these vesicles. An alternative method for removal of contaminant high-abundant proteins was developed which preserves the activity of vesicular proteins. Moreover, lipid-affinity and lectin-affinity-based novel methods to enrich membrane vesicles from minimally processed urine were evaluated and developed. More than 600 proteins were identified in urinary membrane vesicles using shotgun proteomic analysis. Post-translational modification (PTM) proteomics was carried out to identify the PTM status of vesicular proteins. Many different PTMs like glycosylation, ubiquitination and palmitoylation were assessed. Surface glycan profiles of these vesicles were elucidated using fluorophore-linked lectin assay (FLLA) employing 18 different lectins. Lectin blotting, lectin-affinity chromatography using multiple lectins and hydrazide chemistry based enrichment of glycoproteins were carried out. As a result, 108 glycoproteins were identified. Immuno-affinity chromatography was used to enrich and identify ubiquitin-conjugated proteins present in urinary membrane vesicles. A number of potential palmitoylated proteins were identified as well. Computational prediction and validation methods were applied to these protein lists. In conclusion, novel methods to isolate urinary membrane vesicles were developed. In addition, a thorough proteomic characterisation of contents of urinary membrane vesicles was achieved. This work will serve as platform for further characterization of urinary membrane vesicles.