The majority of fluorescence imaging techniques make considerable use of fluorescent dyes. Nevertheless, many structural modification techniques currently in use could not adjust the fluorophores′ properties (such as brightness, photostability, water solubility, and permeability). Herein, we report azaspiroketal‐based auxochrome on naphthalimide and nitrobenzoxadiazole dyes. Their photophysical parameters revealed higher brightness and solubility than their azetidine counterparts in aqueous solution. Computational experiments were performed to study the electronic effect of azetidine and azaspiroketal on both dyes. Cytotoxicity assay indicated that azaspiroketal‐based fluorophores are safe to use for their biological application. The photobleaching studies and successful application of new dyes in bioimaging were demonstrated in MDA‐MB‐231 cell lines.