A new antibody-based protein array assay is described. This assay combines the advantages of the specificity of enzyme-linked immunosorbent assays (ELISA), sensitivity of enhanced chemiluminescence (ECL) and high-throughput of microspot. In this system, the capture proteins, either antibodies or antigens are spotted onto membranes in an array format. Biological samples are then incubated with membranes. After antigens or antibodies in the samples bind to their corresponding targets and unbound proteins are washed away, the membranes are exposed to Horseradish Peroxidase (HRP)-conjugated antibody(ies). The signals are finally visualized with ECL system. Experiments demonstrate that multiple cytokines and antibodies can be simultaneously detected using this new approach. The procedure is so simple that no sophisticated equipment is required. The concept should be able to be extended to develop a high-throughput protein array system. Future applications of this new approach include direct protein expression profiling, immunological disease diagnostics and discovery of new biomarkers.