Pancreatic β-cell proliferation has been gaining much attention as a therapeutic target for prevention and treatment of diabetes. In order to evaluate potential β-cell mitogens, accurate and reliable methods for detection and quantification of the β-cell proliferation rate are indispensable. In this study, we developed a novel tool that specifically labels replicating β cells as mVenus+ cells by using RIP-Cre;R26Fucci2aR mice expressing the fluorescent ubiquitination-based cell cycle indicator Fucci2a in β cells. In response to β-cell proliferation stimuli such as insulin receptor antagonist S961 and diet-induced obesity (DIO), the number of EdU+ insulin+ cells per insulin+ cells and the number of mVenus+ cells per mCherry+ mVenus- cells + mCherry- mVenus+ cells were similarly increased in these mice. Three-dimensional imaging of optically cleared pancreas tissue from these mice enabled quantification of replicating β cells in the islets and morphometric analysis of the islets following known mitogenic interventions such as S961, DIO, pregnancy and partial pancreatectomy. Thus, this novel mouse line is a powerful tool for spatiotemporal analysis and quantification of β-cell proliferation in response to mitogenic stimulation.