Cell isolation protocols are of paramount importance for studying cells from a primary context. Long incubation times are likely to influence gene expression, which will confound interpretations on the obtained data. Every organ poses different problems when preparing single cell suspensions. The vasculature poses an addition problem due to the tight association between mural cells and endothelial cells. Here we share a protocol for quick and efficient isolation of vascular cells from mouse lungs. The entire protocol can be completed in 45 minutes. This protocols has been optimized for quick generation of pure single cells from the vasculature, but is also applicable for other cell types and organs .