The extraordinarily stable, non-covalent interaction between avidin and biotin is one of the most commonly exploited tools in chemistry and biology. Methods for derivatization with biotin of a variety of molecules (in particular, proteins) have been introduced, in order to allow their efficient recovery, immobilization and detection with avidin-based reagents. The field has evolved very rapidly and the applications have become more and more sophisticated. Cell surface protein studies have enormously benefited from refinements of this technology. It is now possible to specifically biotinylate one single membrane protein or to fish out a membrane receptor bound to its ligand. The release of biotinylated molecules from the avidin-based reagents, however, may still represent a major problem, due to the stability of the complex. This review will examine the biotin–avidin technology for the study of cell surface proteins, discussing reagents and techniques as well as examples of applications in quantitative proteomics.