A manufacturing method of a glass substrate having through holes includes (i) irradiating at a through hole forming target position on a first surface of the glass substrate with a laser light; and (ii) performing a wet etching treatment on the glass substrate. During the wet etching treatment being performed on the glass substrate, an ultrasonic vibration with a frequency of less than 40 kHz is applied to an etchant over at least a part of the wet etching period, referred to as an ultrasonic vibration application period.