Advances in imaging techniques enable high-resolution three-dimensional (3D) visualization of vascular networks over time and reveal abnormal structural features such as twists and loops, and their quantification is an active area of research. Here, we showcase how topological data analysis, the mathematical field that studies the “shape” of data, can characterize the geometric, spatial, and temporal organization of vascular networks. We propose two topological lenses to study vasculature, which capture inherent multiscale features and vessel connectivity, and surpass the single-scale analysis of existing methods. We analyze images collected using intravital and ultramicroscopy modalities and quantify spatiotemporal variation of twists, loops, and avascular regions (voids) in 3D vascular networks. This topological approach validates and quantifies known qualitative trends such as dynamic changes in tortuosity and loops in response to antibodies that modulate vessel sprouting; furthermore, it quantifies the effect of radiotherapy on vessel architecture.