The potential of antibodies as magic bullets for targeting therapeutic drugs or imaging agents has been well-documented and is now an emerging area of molecular medicine. Because antibodies act first by binding to their specific antigens it is easy to envisage that the specific activity of antibody-based drugs depends to a large extent on the antigen-binding affinity of antibodies. For a variety of reasons under many circumstances instead of whole antibodies, smaller antigen-binding units of antibodies such as Fabs and Fvs are better suited to perform the job of the magic bullet. The conversion of whole antibodies into Fabs and Fvs (scFvs or dsFvs) is often associated with a drastic reduction in the antigen-binding affinity. It is therefore pertinent that for efficient use of Fvs as targeting agents their binding affinities need to be improved. Until the recent past there had been two different approaches to improve binding affinity of antibodies (whole IgGs, Fabs, and Fvs). One approach relied on the high-resolution crystal structure of antibody-antigen complex followed by engineering of key contact residues in the Fv portion to enhance the interaction. Although this approach is very logical and has greater chances of success, it is not readily feasible. The other approach that has been used to improve affinities of antibodies involved mutating the CDRs randomly or semirandomly and create large expression libraries (for example, scFv or Fab phagedisplay libraries), which served as a potential source of high-affinity variants. This approach has been used successfully several times and in fact has become the method of choice to improve antibody affinity. However, it requires the construction, maintenance, and handling of large and/or multiple phage-display libraries. These tasks require technical expertise and at times they can be time-consuming and expensive.