G-quadruplexes (G4s) are non-canonical nucleic acid structures that form in G-rich regions of the genome and threaten genome stability by interfering with DNA replication. However, the underlying mechanisms are poorly understood. We have recently found that G4s can stall eukaryotic replication forks by blocking the progression of replicative DNA helicase, CMG. In this paper, we detail the methodology of DNA unwinding assays to investigate the impact of G4s on CMG progression. The method details the purification of recombinantly expressed CMG from the budding yeast, Saccharomyces cerevisiae, purification of synthetic oligonucleotides, and covers various aspects of DNA substrate preparation, reaction setup and result interpretation. The use of synthetic oligonucleotides provides the advantage of allowing to control the formation of G4 structures in DNA substrates. The methods discussed here can be adapted for the study of other DNA helicases and provide a general template for the assembly of DNA substrates with distinct G4 structures.