This study described the pharmacokinetics of the intravenous fluorophore, fluorescein, and aimed to evaluate its utility for use in upper gastrointestinal confocal endomicroscopy (CEM). Six healthy, mature, mixed-breed dogs were anesthetized and then dosed intravenously with fluorescein at 15 mg/kg. Blood samples were collected at predetermined time-points. Dogs were examined by upper gastrointestinal confocal endomicroscopy and monitored for adverse effects. Plasma fluorescein concentrations were measured using high-performance liquid chromatography (HPLC) with UV/Vis detection. Mean plasma concentration at 5 min was 57.6 ± 18.2 mg/L, and plasma concentrations decreased bi-exponentially thereafter with a mean concentration of 2.5 mg/L ± 1.26 at 120 min. Mean terminal plasma elimination half-life (t½β) was 34.8 ± 8.94 min, and clearance was 9.1 ± 3.0 mL/kg/min. Apparent volume of distribution at steady-state was 0.3 ± 0.06 L/kg. Fluorescein provided optimal fluorescent contrast to enable in vivo histologically equivalent evaluation of topologic mucosal morphology within the first 30 min following intravenous administration. Adverse effects were not observed. Based upon the calculated clearance, a constant rate infusion at a rate of 0.18 mg/kg/min is predicted to be adequate, following an initial loading dose (2 mg/kg), to maintain plasma concentration at 20 mg/L for optimal CEM imaging during the study period.