Urine exosomes (extracellular vesicles; EVs) contain (micro)RNA (miRNA) and protein biomarkers that are useful for the non-invasive diagnosis of various urological diseases. However, the urinary Tamm-Horsfall protein (THP) complex, which forms at reduced temperatures, may affect EV isolation and may also lead to contamination by other molecules including microRNAs (miRNAs). Therefore, we compared the levels of three miRNAs within the purified EV fraction and THP- protein-network. Urine was collected from healthy donors and EVs were isolated by ultracentrifugation (UC), two commercial kits or sepharose size-exclusion chromatography (SEC). SEC enables the separation of EVs from protein-complexes in urine. After UC, the isolation of EV-miRNA was compared with two commercial kits. The EV isolation efficiency was evaluated by measuring the EV protein markers, Alix and TSG101, CD63 by Western blotting, or miR-375, miR-204 and miR-21 by RT-qPCR. By using commercial kits, EV isolation resulted in either low yields or dissimilar miRNA levels. Via SEC, the EVs were separated from the protein-complex fraction. Importantly, a different ratio was observed between the three miRNAs in the protein fraction compared to the EV fraction. Thus, protein-complexes within urine may influence EV-biomarker studies. Therefore, the characterization of the isolated EV fraction is important to obtain reproducible results.