The increasing demand in studying cellular functions in cultured cells under various levels of oxygen and hydrogen peroxide (H2O2) is only partly fulfilled by conventional approaches such as hypoxia chambers, bolus additions of H2O2 or redox-cycling drugs. This article describes the recently developed enzymatic GOX/CAT system consisting of glucose oxidase (GOX) and catalase (CAT) that allows the independent control and maintenance of both H2O2 and hypoxia in cell culture. In contrast to hypoxia chambers, the GOX/CAT system more rapidly induces hypoxia within minutes at a defined rate. The degree of hypoxia is dependent on the GOX activity and the diffusion distance of oxygen from the medium surface to the adherent cells. In contrast, H2O2 levels are solely controlled by the ratio of GOX and CAT activities. They can be adjusted at non-toxic or toxic dosages over 24 hours. Thus, the GOX/CAT system mimics a non-phosphorylating respiratory chain and allows to adjust H2O2 levels under hypoxic conditions truly simulating H2O2 release e.g. by inflammatory cells or intracellular sources. GOX/CAT can be employed to address many questions ranging from redox signaling to ischemia/reperfusion studies in transplantation medicine. Factors such as HIF1 alpha that respond both to hypoxia and H2O2 are an especially attractive target for the novel methodology. Several applications are discussed in detail to demonstrate the technical requirements and potentials. In addition, simplified protocols are presented for cell or molecular biology labs without dedicated biophysical equipment.