When human cells enter mitosis, chromosomes undergo substantial changes in their organisation to resolve sister chromatids and compact chromosomes. Despite the fundamental importance of this phenomenon to genome stability, we still do not fully comprehend the timing and coordination of these events. To address these questions, we need to evaluate the progression of both sister chromatid resolution and chromosome compaction in one assay. We achieved this by analysing changes in configuration of marked chromosome regions over time, with high spatial and temporal resolution. This assay showed that sister chromatid resolution is an iterative process that begins in late G2 phase and completes in prophase. Cohesins and WAPL antagonistically regulate sister chromatid resolution in late G2 and prophase whilst local enrichment of cohesin on chromosomes prevents precocious sister chromatid resolution. Moreover, our assay allowed quantitative evaluation of the timing and efficiency of condensin II and I activities in promoting sister chromatid resolution and chromosome compaction, respectively. Thus, our real-time assay sheds new light on the dynamics of mitotic chromosome resolution and compaction.