Background: There is an urgent need for the development of innovative diagnostic and therapeutic strategies in bladder cancer (BlCa) management, due to its significant incidence, mortality and morbidity. Extracellular vesicles (EVs) represent highly promising candidates as biomarkers, as their cargo is highly protected from degradation and they can be isolated non-invasively from urine. Furthermore, they play a pivotal role in intercellular communication that drives disease progression, making them suitable candidates for tailored therapy approaches. Methods: We employed high-throughput mass-spectrometry to perform a comprehensive proteomic analysis of cell lysates and matched EVs. EVs were isolated from six distinct human BlCa cell lines, representatives of the complete disease spectrum, in order to identify a BlCa-EV specific signature. The translational ability of this signature was analyzed in urinary EV (uEV) samples from BlCa patients and healthy controls. Results: This study established a new lysate-to-EV signature that is common to all malignant cell lines. Afterwards, this profile was analyzed in BlCa uEVs, identifying a robust and novel 13 protein BlCa-specific signature, which includes ITGA2, ITGA6, SCRIB and TFRC. Conclusion: We unveiled a novel, specific panel of EV-derived proteins that have potential to be further applied for future functional studies, non-invasive diagnostic assays and targeted therapeutic interventions against BlCa.