D-Xylitol is found in low content as a natural constituent of many fruits and vegetables. It is a five-carbon sugar polyol and has been used as a food additive and sweetening agent to replace sucrose, especially for non-insulin dependent diabetics. It has multiple beneficial health effects, such as the prevention of dental caries, and acute otitis media. In industry, it has been produced by chemical reduction of D-xylose mainly from photosynthetic biomass hydrolysates. As an alternative method of chemical reduction, biosynthesis of D-xylitol has been focused on the metabolically engineered Saccharomyces cerevisiae and Candida strains. In order to detect D-xylitol in the production processes, several detection methods have been established, such as gas chromatography (GC)-based methods, high performance liquid chromatography (HPLC)-based methods, LC-MS methods, and capillary electrophoresis methods (CE). The advantages and disadvantages of these methods are compared in this review.