The aim of the present study was to develop a spectrophotometric based assay for the determination of binding parameters like binding constant and binding free energy of the free and 1,1-diphenyl-2-picrylhydrazyl (DPPH) bound forms of a set of antioxidant standards (AS). The determination of binding constant and binding free energy is based upon the decrease in absorbance of the electronic absorption spectrum of a 10-4 M acetonitrile solution of DPPH in the presence of a given concentration of a solution of antioxidant standards in the same solvent.