This section introduces a simple, rapid, high-throughput methodology for the site-specific biotinylation of proteins for the purpose of fabricating functional protein arrays. Step-by-step protocols are provided to generate biotinylated proteins using in vitro, in vivo, or cell-free systems, together with useful hints for troubleshooting. In vitro and in vivo biotinylation rely on the chemoselective native chemical ligation (NCL) reaction between the reactive alpha-thioester group at the C-terminus of target proteins, generated via intein-mediated cleavage, and the added cysteine biotin. The cell-free system uses a low concentration of biotin-conjugated puromycin. The biotinylated proteins can be either purified or directly captured from crude cellular lysates onto an avidin-functionalized slide to afford the corresponding protein array. The methods were designed to preserve the activity of the immobilized protein such that the arrays provide a highly miniaturized platform to simultaneously interrogate the functional activities of thousands of proteins. This is of paramount significance, as new applications of microarray technologies continue to emerge, fueling their growth as an essential tool for high-throughput proteomic studies.