In vitro selections performed by phage display are often performed against a recombinant antigen adsorbed on a solid surface. Passive adsorption of the antigen involves its partial denaturation, which can lead to binders able to strongly recognize the adsorbed antigen but displaying a poor affinity for the native antigen. As an alternative, the use of in vitro or in vivo biotinyled antigens avoids such issues, while allowing a precise control of the interaction between the phage particle and the antigen that takes place in solution. The whole complex can be retrieved by a short incubation over a streptavidin-coated solid phase. This chapter provides detailed protocols to perform in vitro or in vivo biotinylation of the antigen and phage display selection and screenings on biotinylated antigens.