Abstract: Galactooligosaccharides (GOSs) are nondigestible oligosaccharides and are comprised of 2 to 20 molecules of galactose and 1 molecule of glucose. They are recognized as important prebiotics for their stimulation of the proliferation of intestinal lactic acid bacteria and bifidobacteria. Therefore, they beneficially affect the host by selectively stimulating the growth and/or activity of a limited number of gastrointestinal microbes (probiotics) that confer health benefits. Prebiotics and probiotics have only recently been recognized as contributors to human health. A GOS can be produced by a series of enzymatic reactions catalyzed by β-galactosidase, where the glycosyl group of one or more D-galactosyl units is transferred onto the D-galactose moiety of lactose, in a process known as transgalactosylation. Microbes can be used as a source for the β-galactosidase enzyme or as agents to produce GOS molecules. Commercial β-galactosidase enzymes also do have a great potential for their use in GOS synthesis. These transgalactosyl reactions, which could find useful application in the dairy as well as the larger food industry, have not been fully exploited. A better understanding of the enzyme reaction as well as improved analytical techniques for GOS measurements are important in achieving this worthwhile objective.