The blood vessels that supply the inner ear form a barrier between the blood and the inner ear fluids to control the exchange of solutes, protein, and water. This barrier, called the blood-labyrinth barrier (BLB) is analogous to the blood-brain barrier (BBB), which plays a critical role in limiting the entry of inflammatory and infectious agents into the central nervous system. We have developed an in vivo method to assess the functional integrity of the BLB by injecting sodium fluorescein into the systemic circulation of mice and measuring the amount of fluorescein that enters perilymph in live animals. In these experiments, perilymph was collected from control and experimental mice in sequential samples taken from the posterior semicircular canal approximately 30 min after systemic fluorescein administration. Perilymph fluorescein concentrations in control mice were compared with perilymph fluorescein concentrations after lipopolysaccharide (LPS) treatment (1 mg/kg IP daily for 2 days). The concentration of perilymphatic fluorescein, normalized to serum fluorescein, was significantly higher in LPS-treated mice compared to controls. In order to assess the contributions of perilymph and endolymph in our inner ear fluid samples, sodium ion concentration of the inner ear fluid was measured using ion-selective electrodes. The sampled fluid from the posterior semicircular canal demonstrated an average sodium concentration of 145 mM, consistent with perilymph. These experiments establish a novel technique to assess the functional integrity of the BLB using quantitative methods and to provide a comparison of the BLB to the BBB.