Simple SummaryPhotosynthetic organisms, such as land plants, evolved to utilize available light and to use its energy to assimilate carbon dioxide and produce carbohydrates. However, the light intensity often exceeds the ability of plants to successfully utilize absorbed energy, thus inducing stress, manifested by an increased radical concentration inside plant cells and disruption of the inner structures, and consequently decreased plant yield. Plants solve this problem by using a mechanism termed non-photochemical quenching, by which they can dissipate the energy not used in photosynthesis. Modern agriculture, however, also involves indoor plant farming. For indoor plant farming LED-based lighting systems, with non-saturating light intensities, are suitable based on their restricted energy consumption. However, the composition of applied light should first be optimized to maximize its utilization. Our study examined the influence of monochromatic LEDs (red, green, and blue) on the photoprotective and photosynthetic properties of tomato plants. We indicate that monochromatic green light could be considered an important component of lighting systems to alleviate energy dissipation, while blue light enhances photosynthetic efficiency. Our study not only proves the crucial importance of spectrum optimization but also provides evidence that different light wavelengths modify photosynthetic and photoprotective properties.Photosynthetic pigments of plants capture light as a source of energy for photosynthesis. However, the amount of energy absorbed often exceeds its utilization, thus causing damage to the photosynthetic apparatus. Plants possess several mechanisms to minimize such risks, including non-photochemical quenching (NPQ), which allows them to dissipate excess excitation energy in the form of harmless heat. However, under non-stressful conditions in indoor farming, it would be favorable to restrict the NPQ activity and increase plant photosynthetic performance by optimizing the light spectrum. Towards this goal, we investigated the dynamics of NPQ, photosynthetic properties, and antioxidant activity in the leaves of tomato plants grown under different light qualities: monochromatic red (R), green (G), or blue (B) light (L) at 80 µmol m−2 s−1 and R:G:B = 1:1:1 (referred to as the white light (WL)) at 120 µmol m−2 s−1. The results confirm that monochromatic BL increased the quantum efficiency of PSII and photosynthetic pigments accumulation. The RL and BL treatments enhanced the NPQ amplitude and showed negative effects on antioxidant enzyme activity. In contrast, plants grown solely under GL or WL presented a lower amplitude of NPQ due to the reduced accumulation of NPQ-related proteins, photosystem II (PSII) subunit S (PsbS), PROTON GRADIENT REGULATION-LIKE1 (PGRL1), cytochrome b6f subunit f (cytf) and violaxanthin de-epoxidase (VDE). Additionally, we noticed that plants grown under GL or RL presented an increased rate of lipid peroxidation. Overall, our results indicate the potential role of GL in lowering the NPQ amplitude, while the role of BL in the RGB spectrum is to ensure photosynthetic performance and photoprotective properties.