Even though the formation of compact cylindrical chromosomes early during mitosis or meiosis is a prerequisite for the successful segregation of eukaryotic genomes, little is known about the molecular basis of this chromosome condensation process. Here, we describe in detail the protocol for a quantitative chromosome condensation assay in fission yeast cells, which is based on precise time-resolved measurements of the distances between two fluorescently labeled positions on the same chromosome. In combination with an automated computational analysis pipeline, this assay enables the study of various candidate proteins for their roles in regulating genome topology during cell divisions.