In rheumatoid arthritis, the profile, number and functional effects of extracellular vesicles (EVs) in synovial fluid have been reported to change with disease. These changes are believed to not only have roles in disease, but may also serve as clinical biomarkers. However, the complexity of synovial fluid presents challenges in the isolation of pure EV populations, since standard ultracentrifugation-based methods co-isolate large amounts of particulate material including non-EV proteins and cause artefactual vesicle aggregation. These limitations are confounders in studying the content and function of EVs in synovial fluid. We have applied a size exclusion chromatography method to purify small EVs, including exosomes and small microvesicles from synovial fluid of arthritis patients. When compared to EV isolation by ultracentrifugation and sucrose density gradient ultracentrifugation, our size exclusion chromatography method results in a higher quality EV enrichment, as shown by western blotting which reveals improved separation of EV markers, such as flotillin-1, TSG101 and annexin A1, from non-EV associated contaminants, including human serum albumin and apolipoprotein A-1. Furthermore, when assessed by transmission electron microscopy a reduction in the amount of non-EV amorphous material was detected in fractions positive for EV markers. In summary, our method separates small EVs from the bulk of non-EV synovial fluid protein that is also pelleted by conventional ultracentrifugation-based techniques, and eliminates the potential of EV aggregation which may occur as a result of high speed ultracentrifugation. Hence, our method will likely be useful in future investigations into the precise contents and roles of synovial fluid EVs in rheumatoid arthritis.