The agriculture sector consumes more than two-thirds of world’s limited freshwater resources. However, only a small part of the water (less than 5%) that is taken up by roots is used for plant growth, while the rest (above 95%) is lost due to transpiration through the stomatal apertures. Therefore, reducing the transpiration of agricultural plants will contribute to the preservation of precious water resources. However, reducing the transpiration rate artificially is difficult because most plants react delicately and negatively, resulting in water-stressed conditions that often cause different physiological disorders. The present study investigated the transpiration light response in tomato plants (Solanum lycopersicum) grown under LED lights and assessed different irradiation techniques’ ability to reduce transpiration and maintained proper plant growth in a controlled environment. Tomato plants were grown in three enclosed hydroponic units under blue (460 nm) and red (630 nm) LEDs inside an air-conditioned glasshouse. The test plants and multiple replicates were grown five consecutive times, and the irradiation intensity (photosynthetic photon flux density (PPFD)), irradiation pattern (simultaneous/alternate irradiation for red/blue LEDs) and LED combination (number/ratio of red/blue LEDs) were changed each time. The plants’ physiological parameters (transpiration, stomatal conductance, stem-diameter, stem height, and number of leaves) and daily transpiration rates were recorded periodically and analyzed. The results show that a typical photoperiod of 12 hours with simultaneous irradiation of red/blue LEDs produced balanced physiological growth for plants in general. However, when normalized against water use efficiency (transpiration), an alternate irradiation pattern (6 hours: blue LED on/off repeatedly for 15-minute intervals + 6 hours: red LED on/off repeatedly for 15-minute intervals) was the most suitable for tomato cultivation in controlled environments.