在生物化学中，生物素化（英语：biotinylation）是指将生物素共价连接到蛋白质、核酸或其它分子上的过程。由于生物素的分子量不大（分子量为244.31），生物素化反应快速、高效且不易被干扰。生物素化的分子能通过生物素与链霉亲和素、亲和素互作，且不受高热、pH、蛋白酶解的影响。生物素与链霉亲和素、亲和素的结合特异且高效，因此这一互作在生物技术的许多领域有着广泛的应用。另外，多个生物素化分子可以发生交联来形成个科研人员感兴趣的蛋白，同时允许和多个链霉亲和素、亲和素、中性亲和素蛋白结合，增加了对此蛋白的检测灵敏度。此外还有大量的生物素化分子的应用有待开发。

In biochemistry, biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecule. Biotinylation is rapid, specific and is unlikely to perturb the natural function of the molecule due to the small size of biotin (MW = 244.31 g/mol). Biotin binds to streptavidin and avidin with an extremely high affinity, fast on-rate, and high specificity, and these interactions are exploited in many areas of biotechnology to isolate biotinylated molecules of interest. Biotin-binding to streptavidin and avidin is resistant to extremes of heat, pH and proteolysis, making capture of biotinylated molecules possible in a wide variety of environments. Also, multiple biotin molecules can be conjugated to a protein of interest, which allows binding of multiple streptavidin, avidin or Neutravidin protein molecules and increases the sensitivity of detection of the protein of interest. There is a large number of biotinylation reagents available that exploit the wide range of possible labelling methods. Due to the strong affinity between biotin and streptavidin, the purification of biotinylated proteins has been a widely used approach to identify protein-protein interactions and post-translational events such as ubiquitylation in molecular biology.