The ability of surface plasmon resonance to precisely measure kinetic binding constants was exploited here to indirectly evaluate the thermodynamic dissociation trimerization constant (K(d)) of a designed chimeric protein, IZN-23, derived from an isoleucine zipper and a portion of the N-terminal helix residues of HIV-1 gp41. The method uses two monoclonal antibodies (mAbs) that display different off-rates when binding the monomeric or trimeric IZN-23. A detailed description of the data analysis strategy employed to unravel the K(d) trimerization constant from the observed off-rate kinetic values is presented, and the potential exploitation of this technique in different fields is highlighted.