The comparative analysis of ultracentrifugation (UC) and polyethylene glycol (PEG)-based precipitation for the isolation of exosomes in gouty arthritis synovial fluid (GASF) is rarely reported, and it is not known whether different isolation methods can influence subsequent cytokine analysis.GA patients were enrolled during a 1-year period from May 2021 to May 2022. Morphology, particle number, size, purity, protein concentration, and biomarker proteins of GASF-derived exosomes in both extraction methods were observed using transmission electron microscopy, nanoparticle tracer analysis, bicinchoninic acid assay, and Western blotting. An ELISA-based assay platform was used to detect the cytokines in exosomes using Meso Scale Discovery.Thirty-two cases of fresh GASF were taken and randomly divided between the UC group (n = 16) and the PEG group (n = 16). Transmission electron microscopy images and nanoparticle tracer analysis results showed round vesicles measuring 100 nm on average. The protein expressions of TSG101, CD63, and CD81 in exosomes of the 2 groups were measured via Western blotting. The number and protein concentration of GASF-derived exosome particles from the PEG group were significantly higher than that of the UC group (P < .001). However, in the purity estimation, the UC group reflected significantly higher exosomes extractability (P < .01). Expression of IL-6 and IL-8 in the GASF-derived exosomes were higher in the UC group (P < .05), showing a median of 3.31 (interquartile range, IQR: 0.84-13.16) pg/mL, and a median of 2.87 (IQR: 0.56-13.17) pg/mL, respectively; moreover, IL-1β was mostly undetectable in the PEG group.The UC method was found to yield exosomes of a higher purity, albeit at a lower quantity but with more abundant inflammatory cytokines; whereas the opposite was the case for the PEG group. The chemical precipitation method might not be suitable in terms of extracting GASF-derived exosomes for inflammation and immunity studies.Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.