Protein microarrays are an emerging technology for studying protein expression profiling and protein functions. However, with the current design approaches, the overall performance of protein microarrays can be compromised by diffusion-limited kinetics. We developed a new protein microarray platform that utilizes a filtration assay with protein microarrays printed on protein-permeable nitrocellulose filter membranes. Compared with protein microarrays assayed with the conventional incubation-shaking method, this new approach overcomes the diffusion limit. We demonstrated that this novel technique can improve the overall reaction kinetic rate by 10-fold, yield a dynamic range of 4 decades, and enhance the assay sensitivity and specificity. Further, using multistacking protein chips, at least 14 chips can be probed simultaneously, with 22400 different reactions in a single assay. The advantages of large fluorescent dyes, such as phycobilisome and quantum dots, can be better exploited using the filtration assay. The potential clinical applications of the filtration-based protein microarrays were demonstrated by detecting carcinoembryonic antigen in human plasma samples.