Glass nanofibers are prospective material, because they have the potential to function as biomedical tissues, optical components, or catalysts. Now, precise control of synthesis method is necessary for a variety of glass nanofiber applications. We found that glass nanofibers were generated from the back surface of a substrate during a drilling experiment using a nanosecond pulsed UV laser. In this report, we investigated the generation process. To understand the process, we set up an optical system for generating nanofibers, which is capable of moving a sample linearly using an XY stage, and monitored around the laser spot using a CCD camera. A non-alkaline, thin glass substrate was irradiated with a laser beam of wavelength 355 nm and pulse width 40 ns. As a result, when the scanning speed and focusing position were favorable, glass nanofibers were generated. According to the in situ observation, microparticles were found on the tip of the nanofibers. Also, the glass substrate was modified in a wider range compared with the laser spot size. Thus, we considered that glass nanofibers were generated when the particles were ejected resulting from local heating. Additionally, glass nanofibers could be generated in combination with a galvano scanning system. The generation of glass nanofibers from the back surface of a substrate is advantageous in terms of their collection owing to the reduced interaction with the laser beam.