Uterine fluid (UF) samples were acquired from healthy cows, cows with clinical (CLE) and subclinical endometritis (SE). The extracellular vesicles (EVs) were isolated using size exclusion chromatography-based method. The enrichment of UF-EVs were confirmed with mass-spectrometry of a sample before and after UF-EV isolation, transmission electron microscopy and nanoparticle tracking analysis. We used LC/MS-MS based quantative shotgun proteomics to compare UF-EV proteomes in healthy cows, cows with SE or CLE acquired at 28 to 35 days postpartum (n = 3 per group).