An accurate estimation of dissociation constants of tetracycline antibiotics in acetonitrile-water binary mixtures is very important for several separation techniques such as liquid chromatography and capillary electrophoresis that use these solvent mixtures. In this study, the pK(a) values of five tetracyclines (tetracycline, oxytetracycline, chlortetracycline, doxycycline and metacycline), have been determined in water and acetonitrile-water binary mixtures (10%, 20% and 30% (v/v)) by potentiometry and liquid chromatography (LC). The obtained results show a similar trend for the pK(a1) values of five tetracycline antibiotics as they increase with increasing concentration of organic modifier, which allows a linear relationship between pK(a1) values and mole fraction of acetonitrile to be obtained. The pK(a) values obtained in aqueous medium have been compared with the values predicted by the SPARC on-line pK(a) calculator.