Chromosome organization undergoes substantial changes during S phase, as parental strands unzip at replication forks to create sister chromatids. Structural Maintenance of Chromosomes (SMC) complexes actively organize chromatin fibers in the wake of the replication fork during a normal S phase, but also in response to replicative stress, thereby influencing replication dynamics. The DNA fiber assay is a relatively simple and cheap technique that allows the study of replication dynamics by sequential incorporation of two nucleotide analogs, followed by DNA spreading and immunodetection of replicated tracks. Here, we present a step-by-step protocol to perform DNA fiber assays in human cell lines, providing a quantitative approach to analyze replication fork progression. As an example, we describe its application in wild-type and SMC5/6 mutant cells, treated or not with hydroxyurea to induce replicative stress. This method can be easily customized to other cell types, alternative strategies for SMC complex inactivation, or treatments that perturb DNA replication. In addition, by modifying culture conditions, this approach can be used to investigate not only fork rate but also fork reversal, fork restart, fork protection, and other aspects of global replication dynamics regulated by SMC activities.