Organic low-molecular-weight dyes with activatable fluorescence are widely used in many analytical, biomedical, pharmaceutical, and environment-sensing applications. The functioning principle of these dyes is based on the cleavage of a caging moiety allocated, in general, at the hydroxyl or amino group that is conjugated with the π-electron system of these dyes. Although a variety of activatable dyes have already been developed, the number of dye classes suitable as the activatable fluorophores is still very limited. Oxonol dyes, which are well-known fluorescent reporters, also contain a π-conjugated hydroxyl group but they have never been explored as activatable dyes and, moreover, synthetic pathways for the hydroxyl group functionalization have yet to be elaborated. In this work, for the first time, oxonol dyes with activatable fluorescence were synthesized and evaluated. The hydroxyl group in these oxonols was functionalized with caging aliphatic propionyl (Pp) and aromatic benzoyl (Bz) groups, and anticancer drug chlorambucil (CLB). Under hydrolytic conditions, the caging groups can be cleaved, which results in a dramatic increase of the fluorescence at ∼645 nm, thus making these dyes attractive candidates for fluorescent drug delivery monitoring and other analytical and biomedical applications.