During tumor growth, the complex composition of vasculature is prone to dynamic changes due to mechanic and biochemical challenges. Perivascular invasion of tumor cells to co-opt existing vasculature, but also formation of de-novo vasculature and other effects on the vascular network, may lead to altered geometric vessel properties as well as changes in vascular network topology, which is defined by vascular multifurcations and connections between vessel segments. The intricate organization and heterogeneity of the vascular network can be analyzed with advanced computational methods to uncover vascular network signatures that may allow differentiating between pathological and physiological vessel regions. Herein, we present a protocol to evaluate vascular heterogeneity in whole vascular networks, using morphological and topological measures. The protocol was developed for single plane illumination microscopy images of mice brain vasculature but can be applied to any vascular network.