The cell cycle is a series of events in the process of one cell giving rise to two daughter cells. The mitotic harvesting method, established by Terasima and Tolmach in the 1960s, causes minimal physiological stress on the cells and achieves a high degree of cell cycle synchrony by collecting only mitotic cells from a cultured cell system. The purpose of the present study is to validate the versatility of the mitotic harvesting method using human cervical cell line HeLa cells expressing Fluorescent Ubiquitination-based Cell Cycle Indicators (FUCCI) and to estimate the cell cycle-dependent changes in radiosensitivity in HeLa–FUCCI cells. The image analysis showed that cell cycle synchrony was maintained for at least 24 hours after mitotic cell collection. Also, the clonogenic assay demonstrated changes in radiosensitivity that were cell cycle dependent. These results indicate that the mitotic harvesting method using FUCCI-expressing cells has high versatility in the field of radiation cell biology.