This paper is aimed to estimate the transmittance of the electronic effects in the fluorescein molecule between the phthalic acid residue and the xanthene moiety. Despite the almost orthogonal orientation of the latter with respect to the rest of the molecule, some influence of substituents in this 9-aryl ring on the dissociation of the hydroxyl group of the hydroxyxanthene cannot be ruled out. In order to reveal this (possible) effect, we blocked the carboxylic group via esterification. The reason of using dimethyl sulfoxide as solvent was the high Hammett’s “rho” constant for phenolic group. The pKa values of eight methyl or ethyl esters of 3'-, 4'-, and 5'-nitro and amino fluoresceins were determined in benzoate and salicylate buffer solutions using the spectrophotometric method. For nitro derivatives, the dissociation constants of the cationic forms of the dyes were also determined in diluted p-toluenesulfonic acid. The study reveals increasing in the pKa by 0.2–0.3 units in the case of amino derivatives, while the nitro group decreases the pKas by 0.2–0.8. Also, the position of the substituent is of importance; the 5'-substituents display the least influence. In addition, the pKa values of methyl and ethyl esters of eosin (2,4,5,7-tetrabromofluorescein) were determined in order to clarify the influence of the alkyl group. The absorption maxima and molar absorptivities of the anions, as well as the pKas in DMSO coincide within the margin of errors.