When studying primary tissues, the cell isolation protocol is of critical importance for assessing cellular behavior and characteristics. The vasculature poses an additional problem due to the tight association between mural cells and endothelial cells. Here we share a protocol for efficient, fast and clean isolation of vascular cells from mouse brain. In optimal conditions, a single cell brain solution can be obtained in 75 minutes. The last 30 minutes, the sample is at 4 °C. This protocols has been optimized for generation of pure single cells from the vasculature, but is also applicable for other cell types.