Liquid–liquid phase separation (LLPS) is an increasingly studied property of biological macromolecules that can give rise to membrane-less compartments (“condensates”) in cells. Analogous to droplets that form after the mixing of oil and water, certain biological macromolecules are also capable of de-mixing from the bulk solution, forming condensates whose contents are partitioned from the rest of the cellular environment. Phase transitions in biology are frequently driven by weak, multivalent protein–protein or protein–nucleic acid interactions, as well as by intrinsically disordered regions (IDRs) of proteins. Fluorescence microscopy is one method commonly used for studying the formation of biological condensates both in vitro and in cellulo. Depending on the system, imaging may be performed with protein alone or with the addition of a nucleic acid ligand or substrate to induce phase separation. Here, we describe a protocol for how to produce dye-labeled samples suitable for studies of in vitro phase separation by a human type II topoisomerase using fluorescence microscopy. The approach does not rely on tags or fusion proteins (which can alter LLPS properties) and is generalizable to any protein with a free, N-terminal primary amine.