Hypoxia chambers are a well developed device for investigating the biochemical effect of tissue under hypoxic conditions. The enclosed design of the chamber is essential to prevent re-oxygenation by external air but also limits access to measurements during experiments, resulting in fewer sampling points or only pre- and post-hypoxia data. The present study implements a non-enclosed hypoxia chamber to provide free entry for manipulating microprobes, which makes frequent sampling during experiments feasible. A steady deoxygenated environment is created directly by supplying nitrogen to the culture medium or indirectly by supplying a pre-prepared deoxygenated medium. Three hypoxia chamber schemes were designed and validated to induce a low oxygen level. In addition, nerve growth factor (NGF)-differentiated PC12 cells were utilized to verify the efficiency of ischemic conditions. Results indicate that the design with a continuous supply of nitrogen into the water-trapped chamber provides the most stable deoxygenated environment and prevents the dehydration effect caused by nitrogen pumping. The designed hypoxia chamber can be used for continuous monitoring of optical or electrical properties of neuronal cells insulted by ischemia for drug screening or fundamental research.