Regulatory agencies establish that a broad physicochemical and biological characterization is necessary for the evaluation of comparability between a biosimilar candidate product and a reference commercial drug. Between them, conformational characterization of proteins is of vital importance to determine its folding and biological functions. In this work, the conformational features of a novel monoclonal antibody (called 5G4) were evaluated by means of circular dichroism spectroscopy and fluorescence. Secondary structure and thermal stability of mAbs were determined by circular dichroism in the far ultraviolet, while three-dimensional folding of proteins was analyzed by both circular dichroism in the near ultraviolet and intrinsic tryptophan fluorescence. In all experiments, Herceptin (Roche) was used as control. Both antibodies showed a composition of secondary structure predominantly of β-sheets (55–56%) and thermal stability of ~ 75°C, suggesting structural similarity. The three-dimensional folding of proteins was also similar due to the absorption spectra of the aromatic residues and the emission wavelength maxima by fluorescence were comparable. The values of the fluorescence attenuation constant (Stern-Volmer constant) for increasing concentrations of acrylamide were also similar, suggesting a degree of exposure of tryptophan residues similar, although it was slightly decreased for Herceptin. Our data permit to consider that 5G4 monoclonal antibody showed similar conformational characteristics when compared with Herceptin.