Homologous recombination (HR) is a universally conserved DNA double-strand break repair pathway. Single-molecule fluorescence imaging approaches have revealed new mechanistic insights into nearly all aspects of HR. These methods are especially suited for studying protein complexes because multicolor fluorescent imaging can parse out subassemblies and transient intermediates that associate with the DNA substrates on the millisecond to hour timescales. However, acquiring single-molecule datasets remains challenging because most of these approaches are designed to measure one molecular reaction at a time. The DNA curtains platform facilitates high-throughput single-molecule imaging by organizing arrays of DNA molecules on the surface of a microfluidic flowcell. Here, we describe a second-generation UV lithography-based protocol for fabricating flowcells for DNA curtains. This protocol greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large datasets from individual single-molecule experiments. Drawing on our recent studies of human HR, we also provide an overview of how DNA curtains can be used for observing facilitated protein diffusion, processive enzyme translocation, and nucleoprotein filament dynamics on single-stranded DNA. Together, these protocols and case studies form a comprehensive introduction for other researchers that may want to adapt DNA curtains for high-throughput single-molecule studies of DNA replication, transcription, and repair.