A hypoxic microenvironment effects various signaling pathways in the human body, including those that are critical for normal physiology and those that support tumorigenesis or cancer progression. A hypoxic tumor microenvironment, in particular, modulates cell migration, invasion and resistance to radiotherapy and chemotherapy. Development of the mass spectrometry (MS) technique has allowed for expansion of proteomic study to a wide variety of fields, with the study of tumor hypoxia being among the latest to enjoy its benefits. In such studies, changes in the proteome of tumor tissue or cells induced by the hypoxic conditions are analyzed. A multitude of hypoxic regulatory proteins have already been identified, increasing our understanding of the mechanisms underlying tumor occurrence and development and representing candidate reference markers for tumor diagnosis and therapy. The present review provides the first summary of the collective studies on tumor microenvironment hypoxia that have been completed using MS-based proteomic techniques, providing a systematic discussion of the benefits and current challenges of the various applications.