The stepwise proton-ligand stability constants of thiobarbituric acid (TBA) anion were determined in an aqueous solution via pH-potentiometry and near UV-region spectrophotometry at ionic strength I = 0,1 and temperature T = 20 °C (25 °C). The values (presented in the logarithmic form) obtained by these methods show satisfactory convergence, namely, 2,30 ± 0,01 (20 °C), 2,16 ± 0,01 (25 °C) and 2,37 ± 0,06 (20 °C), respectively. Additionally, the molecular absorption spectroscopy results have shown that thiobarbituric acid could exist in the two predominant forms within pH of 1.1 – 9.2. This latter fact correlates with the conclusions on the TBA diagrams for the acid’s equilibrium particle yield as a function of the aqueous solution pH. Quantum chemical calculations have confirmed that the keto form is more thermodynamically stable than the enol one. The obtained values ​for certain geometric parameters and for the effective charges on Mulliken atoms make it possible to describe the structure of this tautomer and to argue that the acid mainly functions as an N, O-donor ligand during the complexation with metal ions. In addition, the presence of >NH, >C=O, –OH and >C=S functional groups in TBA was qualitatively confirmed via IR-spectroscopy.