In this study, the values of ionization/protonation constants (pK(a)) of pemetrexed and raltitrexed, which are folic acid antimetabolites, were determined by reversed-phase liquid chromatography (RPLC) and UV-visible spectrophotometric methods at 25 degrees C. The (s)(s)pK(a) values were evaluated by retention time (t(R)) in binary acetonitrile-water mixtures with acetonitrile (ACN) contents of 13%, 15%, 17% and 20% (v/v) for the RPLC method and 13%, 15% and 17% (v/v) for the spectrophotometric method. The aqueous pK(a) values ((w)(w)pK(a)) of the studied compounds were calculated from the (s)(s)pK(a) value using the macroscopic parameters (mole fraction and dielectric constant), which play an important role in solvent properties. In addition, the degree of ionization of pemetrexed and raltitrexed was calculated using the (w)(w)pK(a) values.