Camelid single-domain antibody fragments ("nanobodies") provide the remarkable specificity of antibodies within a single immunoglobulin V HH domain. This unique feature enables applications ranging from their use as biochemical tools to therapeutic agents. Virtually all nanobodies reported to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we developed a fully in vitro platform for nanobody discovery based on yeast surface display of a synthetic nanobody scaffold. This platform provides a facile and cost-effective method for rapidly isolating nanobodies targeting a diverse range of antigens. We provide a blueprint for identifying nanobodies starting from both purified and non-purified antigens, and in addition, we demonstrate application of the platform to discover rare conformationally-selective nanobodies to a lipid flippase and a G protein-coupled receptor. To facilitate broad deployment of this platform, we have made the library and all associated protocols publicly available. IntroductionAntibodies have had a transformative impact on science and medicine due to their exceptional specificity and biochemical versatility, enabling applications in almost every aspect of biomedical inquiry. Conventional antibodies are composed of two heavy chains and two light chains. Each chain contributes to antigen binding specificity through a variable domain, termed V H and V L for the heavy and light chain, respectively. A key exception to this general architecture is found in camelids (llamas, alpacas, and their relatives), which possess a parallel antibody repertoire composed solely of heavy chains 1,2 . Such antibodies bind to their target antigens through a single variable domain, termed V HH , which contains the entire antigen-binding surface. Unlike the antigen binding fragments of conventional antibodies (Fabs), isolated V HH domains (also called "nanobodies") can be readily expressed in bacteria as the product of a single gene, and in many cases these fragments can even fold and retain antigen specificity in the reducing environment of the cytosol. Owing to their versatility, nanobodies have found applications in protein biochemistry and structural biology, cell biology, and as potential diagnostic and therapeutic agents [2][3][4][5][6] .