Objective The pathophysiological process of acute high-altitude hypoxia-induced lung injury affects protein expression levels, which are mainly evaluated by Western blot. No systematic study has investigated changes in internal control proteins as calibration loading amounts. Methods Lung injury at an altitude of 6000 m was induced in a low-pressure, low-oxygen chamber for 8, 24, and 72 h using C57BL / 6J mice. Establishment of the model was confirmed by hematoxylin and eosin staining. Expression levels of various internal control proteins,including vinculin, α-tubulin, eukaryotic translation initiation factor 5 ( EIF5), β-actin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected by Western blot, and total protein expression was detected by Coomassie blue staining. Furthermore,the lung injury model in vitro was establised by using,Bronchial epithelial cell (BZAS-2B) andhunman umbilical vein endothelial cells (HUVECS) confirmed by TUNEL staining. Expression levels of internal control proteins were detected by Western blot, and total protein expression was detected by Coomassie Blue staining. Results Acute 8, 24, and 72 h hypoxic models were successfully established in lung tissue, demonstrating consistent total protein expression and stable levels of the internal reference proteins vinculin, α-tubulin, EIF5, and β-actin. GAPDH expression was elevated in the HH8 h, HH24 h, and HH72 h groups compared with the normoxia (Nor) group, but only the increase at HH72 h groups was significant. Similarly, 8, 24, and 48 h hypoxic models were successfully established in BEAS-2B cells and HUVECs, with consistent total protein expression. In BEAS-2B cells, expression levels of the internal reference proteins β-actin and GAPDH were consistent with the normoxic control(NC)group, while vinculin, α-tubulin, and EIF5 expression levels were significantly reduced under hypoxic conditions for up to 24 h. In HUVECs, vinculin and α-tubulin expression levels were also consistent with the NC group, while EIF5, β-actin, and GAPDH expression levels were significantly reduced at 8 h and increased at 48 h. Conclusions Acute hypoxia induces lung tissue injury, and protein expression levels of the internal reference proteins vinculin, α-tubulin, EIF5, and β-actin are stable, making them suitable internal references for Western blot. Additionally, Western blot detected differential expression levels of the internal reference proteins vinculin, α-tubulin, EIF5, β-actin, and GAPDH in BEAS-2B cells and HUVECs, as the most important in vitro lung tissue models of hypoxia-induced injury.