Water stress is the main factor responsible for decreased productivity, which affects the growth and development of crops. Plants respond to stress by accumulating compatible solutes, which have a key role in osmotic adjustment, thereby resulting in osmoprotection of the plants. The loss of water can increase the concentration of compatible osmolytes and molecules that regulate the plant metabolism. These solutes can be metabolized as sugars (sucrose, fructose, trehalosa), amino acids (proline), an amphoteric quaternary amine (glycine betaine), and other low-molecular-weight metabolites. However, among all these compatible solutes, proline and glycine betaine occur the most. Proline is an amino acid that can accumulate in low concentrations under optimal conditions; however, stress conditions contribute to its increased content. Few data are available regarding the levels of endogenous glycine betaine on Solanaceae, which is considered a nonaccumulator under water deficit conditions. The objective of this research was to evaluate the role of compatible osmolytes, glycine betaine and proline, in Capsicum sp. plants under different water deficit conditions. In this study, the presence of endogenous levels of proline and glycine betaine in two species of pepper (Capsicum chinense var. Genesis and Rex and Capsicum annuum var. Padron) were found. The concentration levels of proline were 362, 292, and 246 μmol·g−1 DW for Genesis, Rex and Padron respectively, and irrigation conditions (rehydration) of proline levels increased to 381, 395, and 383 μmol·g−1 DW at 21 days. However, glycine betaine levels were 30–70 μmol·g−1 DW. The relative water content, electrolyte leakage, and soil water potential were also analyzed; therefore, the information suggests that proline contributes better to tolerance to water deficit in the genus Capsicum after 14 days of water deficit treatment. It seems that the contribution of glycine betaine is less effective than that of proline; therefore, it does not have an important role in osmotic adjustment.