Cerebral vasculature is critical in maintaining brain health, as its dysfunction is linked to various neurological illnesses including dementia and stroke. Proteomic studies are integral in understanding the biological mechanism underlying this dysfunction, identifying potential therapeutic targets and novel biomarkers of disease. However, translation of research findings into clinical practice relies on the validity and reproducibility of its methods. Thus, we have developed a novel protocol for the extraction of human cerebral vasculature which is highly compatible with downstream proteomic analyses. This protocol negates the need for bovine serum albumin, a ubiquitous reagent in vessel isolation protocols, yet an undesirable contaminant in proteomics.