The presented study describes the development of reversed-phase liquid chromatography method using a core-shell particle column with a pentafluorophenyl stationary phase for the dissociation constant (pK(a)) determination of the tetracycline group antibiotics (tetracycline, oxytetracycline, chlortetracycline) and their epimers (4-epitetracycline, 4-epioxytetracycline, 4-epichlortetracycline). The pH values were measured in the acetonitrile (ACN)-water binary mixtures, used as mobile phases, instead of in water and take into account the effect of the activity coefficients. Thermodynamic acid dissociation constant (pK(a1)) values of studied antibiotics and their epimers were calculated using retention factor (k) at different mobile phase pH values in studied binary mixtures with ACN percentages of 20, 25, 30 and 35% (v/v). Experimental data were analyzed by using an Origin 7.0 program to fit experimental data to the nonlinear expression derived. From calculated pKa1 values, the aqueous pKa values of studied compounds were calculated by different approaches and these values were compared.