Nondigestible oligosaccharides (NDOs), namely prebiotic oligosaccharides, are functional food ingredients that possess properties that are beneficial to the health of consumers. These include noncariogenicity, a low calorific value, and the ability to stimulate the growth of beneficial bacteria in the colon. Fructooligosaccharides (FOS) represent one of the major classes of oligosaccharides in terms of their production volume. They are manufactured by two different processes, which result in slightly different end products. First, FOS are produced by controlled hydrolysis of inulin. Secondly, FOS can be produced from sucrose by using the transfructosylat-ing activity of the enzyme β-fructofuranosidase (EC 22.214.171.124) at high concentrations of the starting material. This chapter summarizes the methods used for the detection and characterization of enzymes of fungal origin with fructosyltransferase activity and the analytical methods utilized to identify the oligosaccharides produced.