We have previously utilized an M13 phage expression system and codon-based mutagenesis to increase the avidity of the tumor-specific antibody, BR96, 65-fold (Yeltonet al.,1995,J. Immunol.155, 1994–2004). Mutants with improved affinity were identified by screening phage-expressed antibodies on a carcinoma cell line. In this study we describe a more broadly applicable assay which permits rapid and quantitative comparison of affinities of related antibodies produced in an M13 phage expression system. BR96 variants displaying a range of affinities were expressed as soluble antibody fragments (Fabs) in the periplasmic space of bacteria and isolated from small-scale cultures grown in a 96-well format, yielding between 142 ng and 1.06 μg of Fab. Although the small-scale cultures expressed variable levels of Fab, the lower quantities were sufficient to saturate microtiter plates coated with a limiting amount of anti-human Fab antibody, resulting in the capture of uniform quantities of the Fab variants. The relative affinities of the variants were then compared by assessing binding to biotinylated antigen followed by detection with streptavidin–alkaline phosphatase conjugates. This approach permitted the direct comparison of the relative affinities of large numbers of antibody variants in a single step without multiple antibody dilutions. The assay is readily adaptable for screening phage-expressed antibody libraries against any biotinylated target and does not require purified antigen. For instance, the biotinylated BR96 antigen utilized in these studies was from a total cell extract prepared by labeling the surface of live tumor cells followed by detergent extraction. Thus, this approach may be applicable to the screening of antibody libraries against other cell surface antigens, such as transmembrane receptors, which are difficult to purify.