Single-molecule techniques allow researchers to investigate individual molecules and obtain unprecedented details of the heterogeneous nature of biological entities. They play instrumental roles in studying DNA-protein interactions due to the ability to visualize DNA or proteins and to manipulate individual DNA molecules by applying force or torque. Here, we describe single-molecule DNA-flow stretching assays as hybrid tools that combine forces with fluorescence. We also review how widely these assays are utilized in elucidating working mechanisms of DNA-binding proteins. Additionally, we provide a brief explanation of various efforts to prepare DNA substrates with desired internal protein-binding sequences. More complicated needs for DNA-protein interaction research have led to improvements in single-molecule DNA flow-stretching techniques. Several DNA flow-stretching variants such as DNA curtain, DNA motion capture assays, and protein-induced fluorescence enhancement (PIFE) are introduced in this mini review. Singlemolecule DNA flow-stretching assays will keep contributing to our understanding of how DNA-binding proteins function due to their multiplexed, versatile, and robust capabilities. [BMB Reports 2025
58(1): 41-51].