Cohesin is a ring-shaped protein complex that mediates sister chromatid cohesion during cell division. Recently, cohesin was found to build higher-order chromatin structures during interphase, such as topologically associating domains (TADs), loop domains, and chromatin domains. How cohesin formed these structures remained unclear until the development of single-molecule assays that assess the behavior of individual cohesin and DNA molecules. The DNA loop extrusion assay revealed that cohesin can compact DNA by extruding DNA loops. The translocation assay revealed that cohesin can move along DNA. These single-molecule assays are powerful techniques and help our understanding of how higher-order chromosome structures are formed. Here, we describe the DNA loop extrusion assay and the translocation assay by using recombinant human cohesin complex and λDNA to reconstitute the activity of cohesin on DNA at a single-molecule resolution.