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Vascular networks are fundamental components of biological systems. Quantitative analysis and observation of the features of these networks can improve our understanding of their roles in health and disease. Recent advancements in imaging technologies have enabled the generation of large-scale vasculature datasets, but barriers to analyzing these datasets remain. Modern analysis options are mainly limited to paid applications or open-source terminal-based software that requires programming knowledge with high learning curves. Here, we describe VesselVio, an open-source application developed to analyze and visualize pre-binarized vasculature datasets and pre-constructed vascular graphs. Vasculature datasets and graphs can be loaded with annotations and processed with custom parameters. Here, the program is tested on ground-truth datasets and is compared with current pipelines. The utility of VesselVio is demonstrated by the analysis of multiple formats of 2D and 3D datasets acquired with several imaging modalities, including annotated mouse whole-brain vasculature volumes., (© 2022 The Author(s).)