Analysis of thermodynamically rigorous binding isotherms provides fundamental information about the energetics of the ligandmacromolecule interactions and often an invaluable insight about the structure of the formed complexes. The Macromolecular Competition Titration (MCT) method enables one to quantitatively obtain interaction parameters of proteinnucleic acid interactions, which may not be available by other methods, particularly for the unmodified long polymer lattices and specific nucleic acid substrates, if the binding is not accompanied by adequate spectroscopic signal changes. The method can be applied using different fluorescent nucleic acids or fluorophores, although the etheno-derivatives of nucleic acid are especially suitable as they are relatively easy to prepare, have significant blue fluorescence, their excitation band lies far from the protein absorption spectrum, and the modification eliminates the possibility of base pairing with other nucleic acids. The MCT method is not limited to the specific size of the reference nucleic acid. Particularly, a simple analysis of the competition titration experiments is described in which the fluorescent, short fragment of nucleic acid, spanning the exact site-size of the proteinnucleic acid complex, and binding with only a 1:1 stoichiometry to the protein, is used as a reference macromolecule. Although the MCT method is predominantly discussed as applied to studying proteinnucleic acid interactions, it can generally be applied to any ligandmacromolecule system by monitoring the association reaction using the spectroscopic signal originating from the reference macromolecule in the presence of the competing macromolecule, whose interaction parameters with the ligand are to be determined.