Extracellular vesicles (EVs) produced by probiotic bacteria are increasingly recognized as key mediators of host-microbe communication. However, the molecular composition and biological interpretation of bacterial EV proteomes are strongly influenced by the isolation method used. Here, we systematically compared ultracentrifugation (UC) and size exclusion chromatography (SEC) for isolating EVs from Lactobacillus reuteri DSM 20016 and evaluated their effects on vesicle yield, purity, and proteomic profiles. Although UC generated a significantly higher EV yield than SEC, it resulted in substantially lower purity, as indicated by elevated protein contamination and a reduced particle-to-protein ratio. In contrast, SEC improved EV purity by approximately 6.45-fold, demonstrating more effective removal of non-vesicular proteins. Quantitative proteomics identified 982 proteins in bacteria, 677 in UC-EVs, and 861 in SEC-EVs. UC-EVs were predominantly enriched in cytosolic metabolic enzymes, ribosomal proteins, and macromolecular complex-associated components, consistent with co-sedimentation artifacts during high-speed UC. However, SEC-EVs showed selective enrichment of membrane-associated and cell wall-modifying proteins, including peptidoglycan hydrolases, lipoproteins, holin, lytic transglycosylase, penicillin-binding protein, and sulfatase, supporting their origin from regulated envelope remodeling processes. Notably, SEC-EVs contained several proteins including NLP/P60 protein, peptidoglycan hydrolases, EPS biosynthesis-related enzymes, and lipoproteins implicated in anti-inflammatory activities. Overall, our findings demonstrate that EV proteomes are highly isolation-dependent and identify SEC as a superior approach for enhancing proteomic specificity and biological interpretability in bacterial EV research.