The development of cellular structures has garnered significant interest in regenerative medicine for the restoration of lost tissues, organs, and blood vessels. Our research group focuses on the construction of cellular architectures by exploiting the tendency of spheroids to coalesce. Understanding the dynamics of spheroid fusion is pivotal for advancing the construction of larger cellular assemblies and developing innovative methodologies for three-dimensional cellular structures. Therefore, the aim of this study was to perform a morphological analysis of spheroid fusion over time. Human mesenchymal stem cells derived from adipose tissue were used to generate spheroids. Fusion experiments were performed on the second and fifth day of culture using a spheroid formation system, with the number of spheroids ranging from two to four. Subsequently, we monitored the spheroid fusion process by time-lapse imaging at 10-min intervals over three days, utilizing a fluorescent microscope that preserved the culture environment. The images acquired facilitated the development of a novel evaluation method based on the changes in neck radius and circumference of the spheroids. A quantitative assessment of these time-dependent morphological changes revealed distinct fusion behaviors that varied according to the fusion time and the number of spheroids involved.