G-quadruplexes (G4s) are non-canonical nucleic acid secondary structures formed by guanine-rich DNA or RNA sequences. These structures play pivotal roles in cellular processes, including DNA replication, transcription, RNA splicing, and protein translation. High-throughput sequencing has significantly advanced the study of G4s by enabling genome-wide mapping and detailed characterization. This review provides a comprehensive overview of current methods for G4 identification using high-throughput sequencing, focusing on key techniques such as G4-seq, G4-ChIP-seq, G4-CUT&Tag, LiveG4ID-seq, G4assess, HepG4-seq, rG4-seq, RT-stop profiling with DMS-m7G footprinting, G4RP-seq, Keth-seq, and SHALIPE-seq. We discuss the principles, advantages, limitations, and applications of these methods, highlighting their contribution to our understanding of G4 biology. The review also emphasizes the need for improved tools to explore the dynamic behavior of G4s, particularly in living organisms.