Osmolytes are small organic molecules dissolved in cellular and extracellular fluids of all forms of living organisms to maintain the cellular volume. However, in addition to their role in volume regulation, osmolytes do play a variety of roles in biological systems including protein folding, protein disaggregation, protein-protein interaction, and protection against highly osmotic environments. Nature has preferably selected the organic osmolytes for these functions over the inorganic ions due to several reasons. The naturally occurring osmolytes belong to four chemical classes, namely, polyols, sugars, amino acids, and methylamines. In certain instances, osmolytes are used to determine the maximum depth up to which a fish can survive in the marine ecosystem. Additionally, methylamine osmolytes like trimethylamine N-oxide, glycerophosphocholine, and betaine are able to counteract the effect of denaturants under both in vivo and in vitro conditions. In fact, organisms use a large amount of different varieties of osmolytes as an adaptation to the environment where they reside. In this chapter, an up-to-date knowledge of the roles of organic osmolytes in regulating the cell volume is being discussed in detail. Future insights in this direction have also been highlighted.