Human ZGRF1 is one of the many helicases that play important roles in the repair of damaged chromosomes by homologous recombination. However, the lack of biochemical characterization of the ZGRF1 protein has hindered the understanding of its function. We have developed a facile protocol to express human ZGRF1 in insect cells and for its purification. We have also tuned biochemical assays by lowering the protein and DNA concentrations to analyze its functions in RAD51-mediated D-loop formation and dissociation. For complete details on the use and execution of this protocol, please refer to Brannvoll et al. (2020).