In this paper, the nitration of a hydroxyxanthene dye sulfonefluorescein is reported. The orange dyes thus obtained are identified as 4, 5-dinitro and (probably) 2, 4, 5, 7-tetranitro sulfonefluoresceins. The spectral and acid-base properties, interaction with lysozyme, and behavior in surfactant solutions are examined using visible spectroscopy, LDI-ToF, and MALDI-ToF. The xanthene moiety of the dyes is stable against protonation, whereas at high pH values created by NaOH, the rupture of the pyrone cycle readily occurs. Further nucleophilic attack on the central carbon atom results in formation of the carbinolic structure.