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Treatment for inner ear regeneration and protection needs local injection of steroid or new drug for inner ear regeneration into the round window membrane (RWM) in cochlea, but a systemic injection is not available due to its systemic side effects. However, pharmacokinetics of therapeutic agents or steroid locally injected into the inner ear is not well known. Hence, we introduce a new method for the real-time observation of drug delivery in transgenic animals in vivo. We exemplify mice which contain a firefly luciferase (FLuc) gene expression cassette regulated by the murine glial fibrillary acidic protein (GFAP) promoter. Luciferin delivered into the inner ear of those mice reacts with FLuc that is expressed in the GFAP-expressing cells in the cochlear nerve and spiral ganglion, and the resulting bioluminescence is detected by a camera. Using this system, we show the imaging of pharmacokinetic differences between local and systemic (intravenous and subcutaneous) injections of the inner ear.