To provide a method of manufacturing a glass substrate improved in quality with good productivity.

The method of manufacturing the glass substrate has the step of grinding the glass substrate by applying a grinding fluid composition containing a grinding material and water to a gap between a grinding pad and the glass substrate to be ground. The grinding fluid composition has a pH of 4 or less, and the grinding fluid composition and the glass substrate to be ground satisfy the relationship in which a probing depth of a probe of an atomic force microscope, measured by pressing the probe to the surface of the glass substrate to be ground, the glass substrate which is soaked in the grinding fluid composition, is equal to 4 nm or less. Thus the surface roughness of the glass substrate after grinding is reduced, for instance, and grinding exerting an economical grinding speed is achieved.