Antibodies can be successfully engineered and isolated by yeast or phage display of combinatorial libraries. Still, generation of libraries comprising heavy chain as well as light chain diversities is a cumbersome process involving multiple steps. Within this study, we set out to compare the output of yeast display screening of antibody Fab libraries from immunized rodents that were generated by Golden Gate Cloning (GGC) with the conventional three-step method of individual heavy- and light-chain sub-library construction followed by chain combination via yeast mating (YM). We demonstrate that the GGC-based one-step process delivers libraries and antibodies from heavy- and light-chain diversities with similar quality to the traditional method while being significantly less complex and faster. Additionally, we show that this method can also be used to successfully screen and isolate chimeric chicken/human antibodies following avian immunization.