Vascular networks can dictate and indicate states of health and disease. Structural analyses of these networks can facilitate improved understanding of disease states. Recent advances in preclinical imaging techniques and segmentation software have led to the generation of large-scale vasculature datasets. However, these advances have not been accompanied by the development of modernized, open-source analysis software packages. Here we describe VesselVio, an application developed to analyze and visualize pre-segmented 2D and 3D vasculature datasets. Vasculature datasets can be loaded and analyzed with custom parameters to extract numerous quantitative whole-network and individual segment features. Visualization of results and accuracy inspections can be conducted using the interactive visualization tool. The utility and compatibility of VesselVio is demonstrated via the analysis of 3D inferior colliculus segmentations from female and male mice as well as the analysis of 2D retinal fundus images of control, glaucomatous, and diabetic retinopathy patients.