Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks (DSBs) in mammalian species. Upon DSB induction, a living cell quickly activates the NHEJ pathway comprising of multiple molecular events. However, it has been difficult to analyze the initial phase of DSB responses in living cells, primarily due to technical limitations. Recent advances in real-time imaging and site-directed DSB induction using laser microbeam allow us to monitor the spatiotemporal dynamics of NHEJ factors in the immediate-early phase after DSB induction. These new approaches, together with the use of cell lines deficient in each essential NHEJ factor, provide novel mechanistic insights into DSB recognition and protein assembly on DSBs in the NHEJ pathway. In this review, we provide an overview of recent progresses in the imaging analyses of the NHEJ core factors. These studies strongly suggest that the NHEJ core factors are pre-assembled into a large complex on DSBs prior to the progression of the biochemical reactions in the NHEJ pathway. Instead of the traditional step-by-step assembly model from the static view of NHEJ, a novel model for dynamic protein assembly in the NHEJ pathway is proposed. This new model provides important mechanistic insights into the protein assembly at DSBs and the regulation of DSB repair.