The chiral separation of enantiomeric couples of three potential A 3 adenosine receptor antagonists: (R/S)-N-(6-(1-phenylethoxy)-2-(propylthio)pyrimidin-4-yl)acetamide (1), (R/ S)-N-(2-(1-phenylethylthio)-6-propoxypyrimidin-4-yl)acetamide (2), and (R/S)-N-(2-(benzylthio)-6-sec-butoxypyrimidin-4-yl)acetamide (3) was achieved by high-performance liquid chromatography (HPLC). Three types of chiroptical spectroscopies, namely, optical rotatory dispersion (ORD), electronic circular dichroism (ECD), and vibrational circular dichroism (VCD), were applied to enantiomeric compounds. Through comparison with Density Functional Theory (DFT) calculations, encompassing extensive conformational analysis, full assignment of the absolute configuration (AC) for the three sets of compounds was obtained. Chirality 28: 434-440, 2016.