In this study, we examined the usefulness of a tissue-clearing technique for the evaluation of the lung distribution of aerosolized drugs. An aerosol formulation of TexasRed dextran (70 kDa), a model compound of drug carrier for aerosolized drugs, was administered intrapulmonarily to mice using a MicroSprayer, and then DyLight 488-conjugated tomato lectin was administered intravenously to visualize general lung structure via the fluorescent labeling of alveolar and bronchial epithelial cells. Tissue clearing followed by laser scanning confocal microscopy enabled the three-dimensional visualization of intrapulmonary TexasRed dextran and the evaluation of its distribution at the alveolar scale without the preparation of thin tissue sections. These findings suggest that tissue-clearing techniques are useful for the evaluation of intrapulmonary distribution and development of pulmonary drug delivery systems.