The acidity constants of Alizarine Red S were determined spectrophotometrically at 25 degrees C and at constant ionic strength 0.1 M (KNO3) in pure water as well as in aqueous media containing variable mole percentages (5-70%) of organic solvents. The organic solvents used were methanol, ethanol, N,N-dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile and dioxan. The acidity constants of all related equilibria are estimated using the whole spectral fitting of the collected data to an established factor analysis model. DATAN program was applied for determining of acidity constants and pure spectra of different form of Alizarine Red S. The obtained results indicated that acidity constants decrease as the content of an organic solvent in the medium increases. There are linear relationship between acidity constants and the mole fraction of various organic solvents in the solvent mixtures. Effect of various solvents on acidity constants and pure spectrum of each component are also discussed.