Fluorescence resonance energy transfer (FRET) is a distance-dependent process by which energy is transferred from an excited donor fluorophore to an acceptor molecule when the donor and acceptor are in close proximity to each other. Depending on the assay design, FRET can provide a real-time measurement of structural integrity and dynamics of biomacromolecules in solution and is particularly suitable for studying G-quadruplex (G4) nucleic acids and their ligand interactions. FRET-based assays are ideally suited for high throughput screening (HTS) methodology because they are simple, sensitive, and easily automated. G4s are stable nucleic acid structures involved in important regulatory roles in gene replication, transcription, and genomic instability. Four-stranded G4s are promising drug targets as these non-canonical structures are enriched in oncogene promoters, 5′ UTRs, and telomeres, and have been linked to regulation of gene expression in cancer and other diseases. Although molecules that bind to G4s, with subsequent influence on gene expression, have been well documented, the identification of new chemical scaffolds that potently and selectively bind to G4s and control specific gene expression are still much less common. Here, we describe a detailed protocol of a FRET-based HTS methodology to identify novel G4 ligands.