In eukaryotes, newly duplicated chromatids are physically linked together until the onset of chromosome segregation. This physical connection, known as sister chromatid cohesion, is mediated by the cohesin complex, a multisubunit ATPase assembly with a closed-ring conformation that topologically entraps DNA. Cohesin is loaded onto chromatin with the help of regulatory proteins prior to DNA replication and then establishes cohesion when it encounters the replication machinery. This process involves the physical tethering of two replicated sister DNAs and the stabilization of cohesin through acetylation. Here, we describe a biochemical assay to assess the response of cohesin to DNA replication using the purified budding yeast proteins.