Aim. To characterize by different approaches the interaction of interferon α–2b (IFN α–2b) with cytidine 5’–monophophosphoric acid (5’–CMP), guanosine 5’–monophophosphoric acid (5’–GMP), uridine 5’– monophophosphoric acid (5’–UMP) and their disodium salts, in the presence and absence of D–mannitol. Methods. Spectroscopy (time–resolved, circular dichroism and fluorescence), isothermal nanocalorimetry. Results. Using IFN α–2b intrinsic fluorescence measurements we established that 5’–GMP and 5’–CMP interact with IFN α–2b stronger than their disodium salts while no difference of affinity was observed for 5’–UMP and its disodium salt. The presence of D–mannitol enhances the observed effects for 5’–GMP and 5’–CMP, but not for their disodium salts and all forms of 5’–UMP. Thermodynamic measurements reveal that the interaction of all tested 5’–ribonucleoside monophosphoric acids with IFN α–2b is exothermic, while of all disodium salt of 5’–ribonucleoside monophosphates is endothermic. The entropy factor TΔS was calculated to be negative in the presence of all 5’–ribonucleoside monophosphoric acids indicating a decrease of randomness in the system and positive in the presence of their disodium salts indicating an opposite effect. Circular dichroism measurements showed that 5’–ribonucleoside monophosphoric acids induce a decrease of the regular alpha helices content and increase the unstructured regions length in INF α–2b. In contrast, their disodium salts make rather stabilizing effect on the secondary structure elements of the protein. Conclusions. 5’–ribonucleoside monophosphoric acids used in this study interact with IFN α–2b in different way compared to their disodium salts. Acid forms of 5’–ribonucleoside monophosphates possess higher affinity to IFN α–2b and destabilize secondary structure elements of this protein. This may restrain a conformational mobility of IFN α–2b and as a consequence modify its functional activity. In contrast, disodium salts of 5’–ribonucleoside monophosphates display a weak affinity to IFN α–2b and stabilize its secondary structure organization.