Plant biostimulants have received increasing attention in recent years because of their positive effects on crop performance and contribution to agro-ecological sustainability. The aim of this study was to determine the influence of betaine and chitin treatments, alone and in combination, on lettuce plants by changes in the morphology and physiology of plants exposed to regulated deficit irrigation (RDI). Plants were subjected to full irrigation (FI; no water deficiency treatment, field capacity >70%) and RDI (field capacity <50%) conditions until the end of each experiment. We recorded plant yield–related traits, net photosynthesis, and water use efficiency (WUE) values weekly for 4 weeks and carried out three individual experiments to assess the efficacy of biostimulant and irrigation treatments. Betaine (0, 50, and 100 mm/plant) was foliar-treated every 2 weeks during Expt. 1, whereas chitin (0, 2, and 4 g/kg) was applied to the soil at the beginning of Expt. 2. We then applied the optimal concentration of each chemical alone or in combination to the plants as Expt. 3. Compared with negative control, the application of 50 mm betaine and 2 g/kg chitin significantly increased leaf area (LA) per plant by 48.5% and 25.6%, respectively. Furthermore, 50 mm betaine and 2 g/kg chitin treatment showed a clearly protective effect in RDI plants, enhancing their total fresh weights by 26.10% and 75.0%, respectively, in comparison with control. Comparing WUEyield and WUEbiomass, chemical-treated plants had higher values than control. Betaine (50 mm) or chitin (2 g/kg) treatments alone significantly elevated LA, fresh shoot weight, total fresh and dry weights, net photosynthesis, and WUE values, and boosted the water stress tolerance of lettuce under RDI compared with controls. However, a combined treatment of 50 mm betaine and 2 g/kg chitin did not increase the levels of all yield traits under RDI compared with individual chemical treatment. Most leaves appeared healthy, green, and had visually less leaf chlorosis when treated with chitin or betaine under RDI compared with untreated plants subjected to RDI. Our study indicates that applying betaine and chitin improves plant performance against water supply limitations and highlights their potential for the sustainable production of lettuce.