Protein microarrays, an emerging class of proteomic technologies, are quickly becoming essential tools for large-scale and high throughput biochemistry and molecular biology. Recent progress has been made in all the key steps of protein microarray fabrication and application, such as the large-scale cloning of expression-ready prokaryotic and eukaryotic ORFs, high throughput protein purification, surface chemistry, protein delivery systems, and detection methods. Two classes of protein microarrays are currently available: analytical and functional protein microarrays. In the case of analytical protein microarrays, well-characterized molecules with specific activity, such as antibodies, peptide-MHC complexes, or lectins, are used as immobilized probes. These arrays have become one of the most powerful multiplexed detection platforms. Functional protein microarrays are being increasingly applied to many areas of biological discovery, including drug target identification/validation and studies of protein interaction, biochemical activity, and immune responses. Great progress has been achieved in both classes of protein microarrays in terms of sensitivity and specificity, and new protein microarray technologies are continuing to emerge. Finally, protein microarrays have found novel applications in both scientific research and clinical diagnostics.