Aim. This study aimed to investigate the impact of cytosine 5’–monophosphate (5’–CMP), guanosine 5’–monophosphate (5’–GMP), and uridine 5’–monophosphate (5’–UMP), along with their disodium salts, in combination with D–mannitol (D–M), on the conformation and fluorescence properties of interferon α–2b (IFN), and to determine the energy parameters governing these interactions. Conclusions. Overall, the interactions involving 5’–nucleoside monophosphate acids, their salts, and IFN α–2b are influenced by a nucleotide type (purine vs. pyrimidine), the presence of mannitol, and chemical form (acid vs. salt). These interactions significantly affect fluorescence quenching, binding affinity, thermodynamics, energy transfer efficiency, and secondary structure alterations of IFN α–2b.