<p>Isoproterenol (ISO) is a sympathomimetic amine, an analog of epinephrine (E) and norepinephrine (NE), characterized by the <i>N</i>-alkyl substitution. The crystallographic structure was optimized by several common functionals (B3LYP, CAM-B3LYP, B3PW91, M05-2X, M06-2X) in conjunction with the 6-311++G(d,p) basis set. The appropriate level of theory was determined by comparing the experimental and theoretical bond lengths and angles. Different isomers of ISO were examined, and their stability interactions were quantified by the Natural Bond Orbital (NBO) and Quantum Theory of Atoms in Molecules (QTAIM) approaches. The experimental spectra (infrared, Raman, NMR, and UV-Vis) were simulated and assigned after the optimization at the M05-2X/6-311++G(d,p) level of theory. The antiradical activity was determined towards two model (DPPH^• and ABTS^•+) and two biologically relevant (HO^• and Asc^•) radical species and compared to the activity of E and NE. The structural features governing activity and preferred mechanism were elucidated by the quantum chemical methods. The Sequential Proton Loss Electron Transfer (SPLET) mechanism was a dominant one both thermodynamically and kinetically. The protein binding affinity towards Bovine Serum Albumin (BSA) was investigated by spectrofluorimetric titration and molecular docking simulations. The most important interactions were outlined. The importance of <i>N</i>-alkyl substitution proved substantial for the interactions with amino acids, but it did not affect the antiradical activity.</p> Graphical abstract <p>Structure, spectra, intramolecular interactions, protein binding affinity and antioxidant capacity of isoproterenol, a sympathomimetic amine, were examined by a combination of experimental and theoretical methods and compared to those of epinephrine and norepinephrine. The importance of ending groups attached to amino nitrogen atoms and the radical scavenging mechanism were discussed.</p>