There are numerous advantages of employing enzymes as catalysts in organic solvents or aqueous solutions containing organic solvents instead of water. A few natural enzymes which are stable in the presence of organic solvents have been discovered. However, almost all natural enzymes are easily denatured and inactivated in the presence of organic solvents. Therefore, several physical and chemical methods, such as immobilization, modification, and entrapment, for stabilizing enzymes in the presence of organic solvents were developed. Protein engineering using site directed mutagenesis and directed evolution are useful for clarifying why organic solvent-stable enzymes are stable in the presence of organic solvents and for developing organic solvent-stable mutant enzymes.