Pulmonary microvascular endothelial cells (ECs) are integral to the alveoli-capillary barrier of the lung. The EC barrier integrity is known to be disrupted in severe lung diseases such as acute respiratory distress syndrome (ARDS), pneumonia and pulmonary edema. Mice are commonly used to model these diseases, dictating an increasingly high demand for murine ECs isolation and culture. Despite the significant number of protocols for the culture of various types of murine cells, the isolation of microvascular endothelial cells remains a challenging procedure. In our manuscript we developed adetailed step-by-step refined method for isolation murine pulmonary microvascular ECs for in vitro studies. We separated cells using platelet endothelial cell adhesion molecule antibody and characterized ECs with antibodies against intercellular adhesion molecule-1, acetylated-low density lipoprotein, and vascular endothelial (VE)-cadherin. Further, we confirmed microvascular origin of these cells using Griffonia simplicifolia and Helix pomatia (negative control) staining. Barrier properties of EC monolayer were characterized by conducting electric cell-substrate impedance sensing experiments with the edemagenic agents, lipopolysaccharide and nocodazole, and known barrier-protective agents, adenosine and sphingosine-1-phosphate. The described complete protocol provided consistent and reproducible results.