Chromophore-assisted light inactivation (CALI) is a potentially powerful tool for the acute disruption of a target protein inside living cells with high spatiotemporal resolution. This technology, however, has not been widely utilized, mainly because of the lack of an efficient chromophore as the photosensitizing agent for singlet oxygen (O-1(2)) generation and the difficulty of covalently labeling the target protein with the chromophore. Here we choose eosin as the photosensitizing chromophore showing 11-fold more production of O-1(2) than fluorescein and about 5-fold efficiency in CALI of beta-galactosidase by using an eosin labeled anti-beta-galactosidase antibody compared With the fluorescein labeled one To covalently label target protein with eosin, we synthesize a membrane permeable eosin ligand for HaloTag technology, demonstrating easy labeling and efficient inactivation of HaloTag-fused PKC-gamma and aurora B in living cells. These antibody- and HaloTag-based CALI techniques using eosin promise effective biomolecule inactivation that is applicable to many cell biological assays in living cells.