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Blue light and ultra-violet (UV) light have been shown to influence plant growth, morphology, and quality. In this study, we investigated the effects of pre-harvest supplemental lighting using UV-A and blue (UV-A/Blue) light and red and blue (RB) light on growth and nutritional quality of lettuce grown hydroponically in two greenhouse experiments. The RB spectrum was applied pre-harvest for two days or nights, while the UV-A/Blue spectrum was applied pre-harvest for two or four days or nights. All pre-harvest supplemental lighting treatments had a same duration of 12 h with a photon flux density (PFD) of 171 μmol m⁻² s⁻¹. Results of both experiments showed that pre-harvest supplemental lighting using UV A/Blue or RB light can increase the growth and nutritional quality of lettuce grown hydroponically. The enhancement of lettuce growth and nutritional quality by the pre-harvest supplemental lighting was more effective under low daily light integral (DLI) compared to a high DLI and tended to be more effective when applied during the night, regardless of spectrum.