Here we describe a detailed protocol for the one-step preparation of antigen-specific human chimeric immunoglobulin G (IgG) monoclonal antibodies (mAbs) using an in vitro antibody design method referred to as the ADLib (Autonomously Diversifying Library) system. This method employs a chicken B cell line DT40-based library in which the variable regions of the Ig gene loci have been highly diversified by treatment with the histone deacetylase inhibitors. DT40 cells express both membrane-bound and secreted forms of chicken IgM. This property allows a rapid screening and selection of antibody-producing B cells from the library by using magnetic beads conjugated with any antigen of interest. To apply the ADLib system to the direct generation of human chimeric antibody, we have inserted a DNA segment coding for the constant region of human IgG into the chicken IgM heavy-chain locus of DT40 cells by homologous gene targeting. By a mechanism of alternative splicing, the resulting DT40 strain simultaneously expresses chimeric human IgG that contain the same Ig variable region sequences as the membrane-bound chicken IgM displayed at the cell surface. Application of the ADLib system to this human Ig-inserted DT40 strain enables the one-step isolation of human chimeric IgG that is specific for any antigen of interest and can be easily purified for immediate use.