AbstractSince its discovery in the first half of the twentieth century, the high‐affinity, noncovalent interaction between biotin (vitamin H) and the avian protein avidin (and its bacterial homologs) has been exploited for many diverse biotechnology applications. This unit provides several basic protocols for labeling various protein reactive groups with biotin. These protocols can be applied not only to labeling in vitro or in tissue culture, but also to in vivo labeling of whole laboratory animals or to ex vivo labeling of surgically resected organs.Curr. Protoc. Protein Sci. 60:3.6.1‐3.6.21. © 2010 by John Wiley & Sons, Inc.