Background and Purpose: D-amino acid oxidase (DAO) has been developed as a chemogenetic tool to precisely manipulate redox levels in tissues and cells by generating H2O2 in the presence of D-amino acids. The enzyme’s significant oxygen consumption during H2O2 production may influence cellular oxygen levels. We set out to explore if DAO activation results in hypoxia, which in turn alters the activity of the cellular oxygen sensors that are prolyl-4-hydroxylase domain enzymes and thus the hypoxia-inducible factor (HIF) pathway. Experimental Approach: We analyzed oxygen consumption rate (OCR), pericellular oxygen concentration, and HIF-1α stabilization in HEK293 cells stably overexpressing DAO in either the cytoplasm or mitochondrial matrix. The obtained experimental data were used to test whether a simple mathematical model can be used to predict oxygen levels. Key Result: Using HEK293 cells expressing DAO in either the cytoplasm or mitochondrial matrix, we found a dose-dependent increase in OCR upon stimulating the DAO reaction, which resulted in a decreased pericellular O2. Correspondingly, HIF-1α protein levels were stabilized over time, reflecting the biological cellular response to DAO-induced hypoxia. Furthermore, we provide a spreadsheet with a simplified diffusion model that allows users to input experimental parameters and assess the risk of hypoxic conditions in cell cultures. Conclusion and Implications: High DAO activity can induce hypoxia due to increased oxygen consumption. Caution should be taken to prevent hypoxic conditions in cell culture experiments while using DAO. The provided spreadsheet is intended to help control oxygen levels in combination with OCR measurements.