High-affinity, highly specific binding proteins are a key class of molecules used in the development of new affinity chromatography methods. Traditionally, antibody-based methods have relied on the use of whole immunoglobulins purified from immune animal sera, from egg yolks, or from murine monoclonal hybridoma supernatants. To accelerate and refine the reagent antibody generation process, we have developed optimized methods that allow the rapid assembly of scFv libraries from chickens immunized with pools of immunogens. These methods allow the simplified generation of a single moderately sized library of single-chain Fv (scFv) and the subsequent isolation of diverse, high-affinity, and high-specificity monoclonals for each individual immunogen, via phage display. Using these methods, antibodies can be derived that exhibit the desired selectivity, such as complete specificity or cross-reactivity to multiple orthologues of the same protein.