The neurovascular niche is crucial for constant blood supply and blood-brain barrier (BBB) function and is altered in a number of different neurological conditions, making this an intensely active field of research. Brain vasculature is unique for its tight association of endothelial cells with astrocytic endfeet processes. Separation of the vascular compartment by centrifugation-based methods confirmed enrichment of astrocytic endfeet processes, making it possible to study the entire vascular niche with such methods. Several centrifugation-based separation protocols are found in the literature; however, with some constraints which limit their applicability and the scope of the studies. Here, we describe and validate a protocol for physically separating the neurovascular niche from the parenchyma, which is optimized for smaller tissue quantities. Using endothelial, neuronal, and astrocyte markers, we show that quantitative Western blot-based target detection can be performed of both the vessel-enriched and parenchymal fractions using as little as a single mouse brain hemisphere. Validation of our protocol in rodent stroke models by detecting changes in tight junction protein expression, serum albumin signals and astrocyte activation, i.e., increased glial fibrillary acidic protein expression, between the ipsilateral and the lesion-free contralateral hemisphere demonstrates this protocol as a new way of detecting BBB breakdown and astrogliosis, respectively.