A new approach for determination the refractive index of a transparent thin film on top of an arbitrary substrate from reflectivity data taken at different layer thickness is proposed. It is shown that the layer refractive index can be found solely from minimum and maximum reflectivity values, without requiring prior knowledge of the substrate’s properties. The substrate can be composed of multiple plane-parallel homogeneous layers with complex refractive indices or may exhibit an arbitrary depth-dependent refractive index profile. These results can also be applied to spectroscopic data but are limited to substrates that are homogeneous and exhibit low refractive index dispersion within the wavelength range used. The proposed theoretical approach is demonstrated by applying it to real experimental reflectivity data from interference fringes images and spectroscopic measurements taken onto a simple substrate and simulated data for a multilayer system.