Drought is an abiotic stress factor that negatively affects plant development and causes economic losses in plant production, and its effect is getting significant every day. Spinach contains a high amount of water and drought stress affects its development as well as causing changes in its biochemical contents. In this study, it is aimed to determine the changes in the morphological, physiological, and biochemical contents of spinach grown under deficit irrigation conditions in different stages of development. For this purpose, by considering five different irrigation levels (I100, I80, I60, I40, and I20 ), and 4 harvest times determined according to the development periods of spinach, a total of 20 experiment units were designed with 3 replications in a completely randomized experimental design. Growth parameters were negatively affected by severe deficit irrigations such as I20 and I40 . While the deficit irrigation caused a decrease in the relative water content (RWC), protein, and chlorophylls a and b, it increased membrane damage and carotenoid content. Furthermore, deficit irrigations have shown significant increases in proline, hydrogen peroxide, malondialdehyde, superoxide dismutase, catalase, and peroxidase. When different irrigation levels and different harvest periods were evaluated together, water use efficiency (WUE) values ​ranged between 2.31 and 5.02 g/L, and the highest WUE value was obtained at the I40 irrigation level and harvest III. As a result of the principal component analysis conducted to evaluate all the data obtained from the trial subjects together, the most important parameters explaining the effect of harvest time and deficit irrigation together were underground fresh weight, number of leaves, hydrogen peroxide, and peroxidase. Consequently, considering the growth parameters, the I100 irrigation level without deficit irrigation and the I80 irrigation level with mild water stress resulted in considerable outputs, especially in the harvests III and IV.