The purpose of this research was to investigate the impact of acute hypoxia on the urinary proteome in a rat model. The experimental design involved placing rats in a hypoxic chamber that simulated conditions at an altitude of 5000 meters for 24 h. Urine samples were collected at three time points: 0, 12, and 24 h post-hypoxia exposure. The urinary proteins were analyzed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In comparison to the control group (prior to hypoxia), a total of 144 differentially expressed proteins were identified in the 12-h hypoxia group, while 129 differentially expressed proteins were noted in the 24-h hypoxia group. Functional annotation analysis indicated that these differentially expressed proteins were associated with various biological pathways pertinent to hypoxic stress, including anti-oxidative stress responses, glycolysis, and the complement and coagulation cascade. The findings of this study suggest that the urinary proteome is capable of reflecting significant alterations in response to acute hypoxic conditions. These results may offer a valuable method for assessing the hypoxic state of the organism and could aid in the detection of hypoxia.