G4 Biology, Volume 695 in the Methods in Enzymology series, highlights high value topics such as Assays to measure G4 resolution by DNA helicases, In gel staining methods of G4 DNA and RNA structures, Methods to probe the folding pathways of four-stranded intercalated cytosine-rich motifs at single base-pair resolution, Analyzing the effect of transcription coupled R-loop formation on G4 formation, Integrated Structural Biology for the study of higher-order G4 structures, Advancing the Study of Protein-G4 Interactions in DNA Repair: Insights from Biolayer Interferometry and Circular Dichroism Spectroscopy, Immunofluorescence microscopy (IF) of G-quadruplex and R-loops, and more. Additional chapters delve into Production of the anti-G-quadruplex antibody BG4 for efficient ChIP-seq and CUT&Tag analysis: from plasmid quality control to validation, Utility of intercalator displacement assays for screening of ligands for i-motif DNA structures, Potentiometric titrations to study ligand interactions with DNA i-motifs, Towards Profiling of the G-Quadruplex Targeting Drugs in the Living Human Cells Using NMR Spectroscopy, and Identification of bona fide G4-binding proteins, Observing Pif1 opening dsDNA and G4 in real time by manipulation under magnetic tweezers, and in vivo detection of DNA secondary structures using Permanganate/S1 Footprinting with direct adapter ligation and sequencing (PDAL-Seq).