Supercoiling is a fundamental property of DNA, generated by polymerases and other DNA-binding proteins as a consequence of separating/bending the DNA double helix. DNA supercoiling plays a key role in gene expression and genome organization, but has proved difficult to study in eukaryotes because of the large, complex and chromatinized genomes. Key approaches to study DNA supercoiling in eukaryotes are (1) centrifugation-based or electrophoresis-based techniques in which supercoiled plasmids extracted from eukaryotic cells form a compacted writhed structure that migrates at a rate proportional to the level of DNA supercoiling; (2) in vivo approaches based on the preferential intercalation of psoralen molecules into under-wound DNA. Here, we outline the principles behind these techniques and discuss key discoveries, which have confirmed the presence and functional potential of unconstrained DNA supercoiling in eukaryotic genomes.