The invention relates to a glass substrate, which comprises a plurality of panel structures and a plurality of test elements, wherein the panel structures are separated from one another; each panel structure comprises a black matrix and a colorful filter layer; black matrixes define a plurality of pixel units on the glass substrate; the colorful filter layers are arranged on the pixel units; the plurality of test elements are positioned at the peripheries of the panel structures, and are provided with a plurality of virtual pixel units; patterns of the virtual pixel units are different from those of the pixel units; and the virtual pixel units are provided with standard defective patterns. In addition, the invention also provides a detection method using the glass substrate and a matrix mask for manufacturing the glass substrate.