Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since EVs are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, they represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation are laborious, time-consuming, and require high inputs. Ghosh and co-workers recently described an isolation method utilizing Heat Shock Protein (HSP)-binding peptide Vn96 to aggregate HSP-decorated EVs, which can be performed at small ‘miniprep’ scale. Based on microscopic, immunoblot, and RNA sequencing analyses this method compared well with ultracentrifugation-mediated EV isolation, but a detailed proteomic comparison was lacking. Therefore, we compared both methods using label-free proteomics of replicate EV isolations from HT-29 cell-conditioned medium. Despite a 30-fold different scale (ultracentrifugation: 60 ml/Vn96-mediated aggregation: 2 ml) both methods yielded comparable numbers of identified proteins (3115/3085), with similar reproducibility of identification (72.5%/75.5%) and spectral count-based quantification (average CV: 31%/27%). EV fractions obtained with either method contained established EV markers and proteins linked to vesicle-related gene ontologies. Thus, Vn96 peptide-mediated aggregation is an advantageous, simple and rapid approach for EV isolation from small biological samples, enabling high-throughput analysis in a biomarker discovery setting.