DNA supercoiling in biological systems can occur via three mechanisms. The first is by the activity of DNA topoisomerases, such as DNA gyrases, that can increase or reduce the linking number of relaxed DNA (Lk0). The second is via DNA translocation motors, such as RNA and DNA polymerases, that produce twin supercoiled DNA domains: one positively supercoiled in front and one negatively supercoiled behind. The third is via molecular interactions that constrain DNA supercoils and thereby produce compensatory unconstrained ones. This chapter describes the use of agarose-gel electrophoresis to detect and quantify the DNA supercoils generated by these mechanisms. Particular emphasis is made on the preparation of a relaxed DNA plasmid as initial substrate that marks the position of Lk0 for calculating ΔLk.