While significant advances have been made in regard to protein microarray surface chemistries, the surface modification strategies developed are generally substrate-specific and cannot be interchanged between different materials and platforms. This current lack of substrate-independent surface modification strategies makes it difficult to compare or transfer fabrication methodologies between dissimilar substrates. To address this shortcoming, we have developed an interchangeable surface scaffold, which can be utilized to fabricate protein microarrays on a variety of materials with nearly identical results. The surface scaffold is deposited by alternate electrostatic assembly of the cationic and anionic polyelectrolytes poly(allylamine hydrochloride) and poly(sodium 4-styrenesulfonate), respectively. Once assembled, the polyelectrolyte scaffold serves to mask the underlying surface properties of different materials and convert them to the surface properties of the scaffold itself. By obtaining common surface properties across different materials, it is possible to eliminate differences in protein surface density, spot diameter, and nonspecific binding of analytes on substrates as diverse as glass, gold, mica, silicon, and polymer. The concept of substrate-independent polyelectrolyte scaffolding described here provides researchers with a powerful new tool that can be utilized to compare, optimize, and transfer useful protein microarray surface chemistries across different materials and platforms.