Phage display is a widely used approach for the rapid isolation of monoclonal antibodies against a wide range of target antigens. The availability of a diverse antibody library is the essential component of any phage display campaign. The generation of an antibody library is coupled with an iterative process termed as biopanning that allows the identification of monoclonal antibodies. The antibody library consists of the antibody repertoire obtained either from human peripheral blood mononuclear cell (PBMC) or directly from sorted B-cell populations. Depending on the classification of the antibody library sought after, the antibody isotype used would differ. Naïve libraries make use of IgM repertoires, whereas immune libraries would apply IgG repertoires as the antibody gene components. The antibody genes are amplified by polymerase chain reaction (PCR) and cloned in a combinatorial fashion to generate a diverse library repertoire. The protocol describes a two-step cloning approach that can be adapted for the construction of either a naïve or immune human antibody library in Fab format. The protocol would also include a standard biopanning protocol using the Fab library.