Evanescent field optical biosensors are label free sensors that measure the variation of the refractive index of the adsorbed layer onto a chip surface and translate this variation into surface concentration of the adsorbed molecule. The evanescent field based techniques depend on a theoretical model of the waveguide to determine the desired parameters of the adsorbed layer. As this layer is not only composed by the biomolecules, but also by some amount of the buffer solution, in this study, we have developed a new calibration method to take into account the refractive index buffer changes. We report a new methodology to characterize each sensor chip before the measurements and we present the refractive indexes of different buffer solutions considering the most common ones used in biosensor applications. This work will set the calibration bases for any optical grating biosensor instrument.