Drought, especially in arid and semi-arid area, has become a major issue for world food security in recent years. Hence, intensive efforts are being made to refrain the negative effects of drought in agricultural cultivation. The application of proline (Pro) and glycine betaine (GB) helps to conserve water and proteins in plant cells and to protect biological membranes under water stress conditions. The present study aimed to reveal the application doses of Pro and GB by applying their increasing doses to spinach and determining the mitigation of adverse effects under water stress conditions. In greenhouse conditions, two different irrigation levels were applied, namely full irrigation (I100) and 50% water stress (I50), according to the factorial experimental design in random plots. In addition, three different Pro doses (1, 2, and 3 µM) and three different GB doses (50, 100, and 150 µM) were applied. It was observed that water stress caused a significant reduction in plant growth parameters. Although Pro and GB doses were not effective under water stress conditions, they contributed to plant growth under full irrigation conditions. Water stress caused a significant increase in catalase (CAT-49%), proline (PL-119%), and protein contents (PT-12%). Pro2, GB1 and GB2 doses caused a significant decrease in CAT, SOD and PL content under water stress conditions. Moreover, higher values were obtained for carotenoid, chlorophyll a, chlorophyll b and total chlorophyll content at the third level dose of Pro and second level dose of GB, and it was observed that some applications of Pro and GB supported plant growth under full irrigation conditions.