Osmolytes such as glutamate, glycine betaine, dimethylsulfoniopropionate, trehalose, trimethylamine N-oxide (TMAO), and hydroxybutyrate protect bacteria from low temperatures, desiccation, urea accumulation, and high salinity. When stress conditions are too extreme for the protective capacity of the osmolyte and proteins are denatured progressively, trehalose stabilizes the polypeptides present in the nonnative state, impairing their aggregation. An intriguing hypothesis concerning the buffering capacity of chemical chaperones suggests that one of the osmolyte crucial roles in vivo would be of assisting protein evolution. The protective effect of osmolytes is not simply quantitative, but qualitative in the sense that each osmolyte seems to buffer specifically a subset of mutations, a feature probably related to the different mechanisms of action displayed by these molecules. Osmolytes belonging to different classes have been used successfully to stabilize purified proteins at both low and high temperatures. Osmolytes can be precious reagents for stabilizing macromolecules.