The authors describe a novel method for the quantitation of differential levels of biomolecules using unlabeled samples and protein-binding arrays for assessing differential expression. Traditional affinity arrays, whether in microplates or protein microarrays, suffer from a few common problems-a shortage of characterized antibodies and highly variable affinities for those available. Also, the assayed proteins could be present in a wide range of concentrations and physicochemical properties, so that it becomes an onerous task to optimize assay conditions for each antibody-antigen pair. Currently, this restricts parallel affinity assays to a low number of carefully selected antibodies and restricts the development of highly multiplexed parallel affinity assays. A displacement strategy allows the use of a much wider range of antibodies, reducing the requirement for matched affinities. The competitive assays described here also show a much higher tolerance for nonspecific background noise. The range of assayed protein concentrations is only limited by the sensitivity of the detection system used. (Journal of Biomolecular Screening 2003:257-263)