Extracellular vesicles (EVs) in urine are a promising source to develop non-invasive biomarkers. However, urine concentration and content are highly variable and dynamic, and actual urine collection and handling often is nonideal, which presents an enormous challenge for urine-based biomarker studies. Furthermore, patients such as those with prostate diseases have challenges in sample collection due to difficulties in holding urine at designated time points. Here, we simulated the actual situation of clinical sample collection to examine the stability of EVs in urine under different circumstances, including urine collection time and temporary storage temperature, as well as the stability of EVs in daily urine sampling under different diet conditions. EVs were isolated by functionalized EVTRAP magnetic beads, and analyzed by nanoparticle tracking analysis (NTA), Western blotting, electron microscopy, and mass spectrometry (MS). EVs in urine remained relatively stable during temporary storage for 6 hours at room temperature and for 12 hours at 4°C, while significant fluctuations were observed in EV amounts from urine samples collected at the different time points from the same individuals, especially under certain diets. Sample normalization with creatinine reduced the coefficient of variation (CV) value among EV samples from 17% to approximately 6%, and facilitated downstream MS analyses. Finally, we based on the results and applied them to screen for potential biomarker panels in prostate cancer by data-independent acquisition (DIA) MS, presenting the recommendation that can facilitate biomarker discovery with nonideal handling conditions.