As natural plant growth stimulators, amino acids are widely used to improve the yield and quality of crops. Several studies have illustrated the effects of different amino acids on lettuce plant parts. However, the effects of applying single amino acids on root growth remain elusive. The objective of this study was to evaluate the effect of root application of L-methionine on the growth of lettuce. In this study, two successive experiments on butterhead lettuce were conducted under hydroponic conditions. Three amino acids, L-methionine (20 mg/L), L-glycine (210 mg/L), and L-tryptophan (220 mg/L), were applied separately. L-methionine significantly increased the growth performance by 23.60%, whereas growth using L-tryptophan and L-glycine decreased by 98.78% and 27.45%, respectively. Considering the results of the first experiment, a second experiment was established with different concentrations of L-methionine (2200 mg/L, 220 mg/L, 22 mg/L, 2.2 mg/L, 0.2 mg/L, and 0.02 mg/L). The plants were allowed to grow for four weeks. Leaf width, plant area, leaf area, chlorophyll contents, etc., were evaluated. The results show that plant growth significantly improved by applying L-methionine at the lowest concentrations of 0.2 mg/L and 0.02 mg/L, which can, therefore, improve hydroponic production of lettuce and, accordingly, human nutrition.