The single truss tomato production system (STTPS) was used to grow tomato plants at a density of 14.3 plants·m⁻² for increasing the tomato yield in Japan. We applied supplemental lighting within the canopy at different tomato development stages to identify the most sensitive stage at which supplemental lighting will most effectively increase yield and quality of tomato fruits. Fluorescent lamps were used to supply intra-canopy lighting to tomato plants (5:00–21:00) during the stages of anthesis (stage 1), rapid fruit development (stage 2), fruit ripening (stage 3), and from initial anthesis to red-ripe fruit (whole stage), respectively. Supplemental lighting applied to tomato plants during stage 2 and whole stage significantly increased the yield and sugar content of tomato fruits. Moreover, the contribution of supplemental lighting to the daily increase of tomato yield was highest at stage 2. The increase of fruit fresh weight and amount of supplemental lighting showed positive linear relationship. Supplemental lighting did not affect the ascorbic acid content of tomato fruits, fruit number per plant, and plant shoot weight among all the treatments. Thus, based on economic advantage, the use of supplemental lighting during the rapid fruit development stage of tomato plants under STTPS was most efficient.