Biotin is a coenzyme for several enzymes that transport carboxyl units in tissue and plays an integral role in gluconeogenesis, lipogenesis, and fatty acid synthesis. Dietary biotin is absorbed in the small intestine, but it is also synthesized in the gut by bacteria. Numerous foods contain biotin, although no food is especially rich (up to 20 g/100 g). The dietary intake of biotin, while low in the neonatal period, increases as newborns switch from colostrum to mature breast milk. Biotin deficiency can be produced by ingestion of large amounts of avidin, found in raw egg whites that bind to biotin. Biotin deficiency has been noted in patients receiving long-term parenteral nutrition and in infants with genetic defects of carboxylase and biotinidase enzymes. The RDA for biotin is 30 g/day. Reference ranges of 200–500 pg/mL have been established in whole blood and serum. Assays had been performed using microbiology functional assay and the Lactobacillus organism. Newer methods of isotopic dilution, chemiluminescent, and photometric assays are now available but rarely used in hospital laboratories. Specimens are usually sent to a reference laboratory for analysis.