A substrate inspection system and substrate inspection method for setting an inspection region having a three-dimensional shape and/or a two-dimensional arbitrary shape as a region of interest on an image of a substrate. The substrate inspection method includes: generating and displaying a 2D image of a substrate based on image data acquired from the substrate having an inspection object; receiving first input information including arbitrary point data or line data for setting a region of interest at a plurality of particular positions of the 2D image from a user; and displaying the region of interest corresponding to the point data or the line data as a 2D region of interest having an arbitrary shape in accordance with the first input information.