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The aim of this study was to investigate the plasma and ocular dexamethasone (DEX) pharmacokinetics characteristics of the sub-tenon microfluidic system (SMS) delivery of dexamethasone sodium phosphate (DEXP) in experimental autoimmune uveitis (EAU) rabbits. The SMS showed sustained DEX release and large amount of drug loading capacity by a miniature pump which connected with a tube that implanted into the sub-Tenon's sac of the EAU rabbit. The DEX concentrations in the plasma and different ocular compartments (cornea, aqueous humor, retina/choroid, sclera and vitreous humor) were quantified by Shimadzu LC–MS 2010 system. The SMS increased total drug delivery of DEX in ocular tissues as compared with traditional combined medication (subconjunctival injection combined with intravenous injection) in EAU rabbit. Moreover, the minimal amount of DEX was found in the blood, indicated that only a small amount of drug could enter into the bloodstream and causing minimal systematic side effect. Overall, the study provided evidence that SMS meet the large amount of drug loading capacity, continuous release drugs and moderate drug level in the EAU ocular tissue and minimal drug level in the blood, which indicated that SMS could be a feasible option for ocular application in clinical trials and maybe can replace or even discard systemic medications for the management of acute ocular inflammation disease.