This study was conducted to provide basic data for high-throughput screening (HTS) system construction based on phenomics. Rice (Oryza sativa cv. Chucheongbyeo) seedlings in vegetative growth stage were grown in the glass house and treated with 0, 3.75, 7.5, 15, and 30% (w/v) of polyethylene glycol (PEG) to give osmotic stress. Three days after PEG treatment, hyper-spectral reflectance images were obtained and analyzed after removing background image in several steps. The reflectance of rice seedlings treated with 15 and 30% of PEG solutions were significantly different at 680 nm, where differences in the chlorophyll reflectance spectrum and visual symptoms were not observed. These results thus indicate that hyper-spectral reflectance observed at 680 nm can be used to screen drought tolerant rice lines. A HTS system equipped with this hyper-spectral reflectance system may play an important role of future rice breeding program.