Solid tumor biopsies are the current standard for precision medicine. However, the procedure is invasive and not always feasible. In contrast, liquid biopsies, such as serum enriched for extracellular vesicles (EVs) represent a non-invasive source of cancer biomarkers. In this study, we compared two EV isolation methods in the context of the protein biomarker detection in inflammatory bowel disease (IBD) and colorectal cancer (CRC). Using serum samples of a healthy cohort as well as CRC and IBD patients, EVs were isolated by ultracentrifugation and ExoQuickTM in parallel. EV associated protein profiles were compared by multiplex-fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and subsequent identification by mass spectrometry. Validation of gelsolin (GSN) was performed using fluorescence-quantitative western blot. 2D-DIGE resolved 936 protein spots in all serum-enriched EVs isolated by ultracentrifugation or ExoQuickTM. Hereof, 93 spots were differently expressed between isolation approaches. Higher levels of GSN in EVs obtained with ExoQuickTM compared to ultracentrifugation were confirmed by western blot (p = 0.0006). Although patient groups were distinguishable after both EV isolation approaches, sample preparation strongly influences EVs' protein profile and thus impacts on inter-study reproducibility, biomarker identification and validation. The results stress the need for strict SOPs in EV research before clinical implementation can be reached.