Single-molecule studies of protein-DNA interactions continue to yield new information on numerous DNA processing pathways. For example, optical microscopy-based techniques permit the real-time observation of proteins that interact with DNA substrates, which in turn allows direct insight into reaction mechanisms. However, these experiments remain technically challenging and are limited by the paucity of stable chromophores and the difficulty of acquiring statistically significant observations. In this protocol, we describe a novel, high-throughput, nanofabricated experimental platform enabling real-time imaging of hundreds of individual protein-DNA complexes over hour timescales.