Adaptation to decreased oxygen availability (hypoxia) is crucial for proper cell function and survival. In metazoans, this is partly achieved through gene transcriptional responses mediated by hypoxia-inducible factors (HIFs). There is abundant evidence that production of reactive oxygen species (ROS) increases during hypoxia, which contributes to the activation of the HIF pathway. In addition to altering the cellular redox balance, leading to oxidative stress, ROS can transduce signals by reversibly modifying the redox state of cysteine residues in certain proteins. Using the “redox fluorescence switch” (RFS), a thiol redox proteomic technique that fluorescently labels reversibly oxidized cysteines, we analyzed endothelial cells subjected to acute hypoxia and subsequent reoxygenation. We observed a general increase in cysteine oxidation during hypoxia, which was reversed by reoxygenation, and two-dimensional electrophoresis revealed the differential oxidation of specific proteins. Using complementary derivatization techniques, we confirmed the modification of individual target proteins and identified specific cysteine residues that were oxidized in hypoxic conditions, thereby overcoming several limitations associated with fluorescence derivatization. These findings provide an important basis for future studies of the role of these modifications in HIF activation and in other acute adaptive responses to hypoxia.