A method of calibrating a substrate positioning system of a lithographic apparatus, the method including: exposing a pattern with the lithographic apparatus on an exposed layer on the surface of a substrate having a reference layer, wherein the pattern corresponds to a movement of the substrate by the substrate positioning system; measuring overlay data between the exposed layer and the reference layer on a plurality of positions on the substrate; transforming the overlay data from a spatial domain to a frequency domain by a discrete cosine transformation; modifying the overlay data in the frequency domain by selecting a subset of the overlay data; transforming the modified overlay data from the frequency domain back to the spatial domain by an inverse discrete cosine transformation; calibrating the substrate positioning system by using the modified overlay data in the spatial domain.