Hypoxia is present in most solid tumours, and is clinically correlated with increased metastasis and poor patient survival. While studies have demonstrated the role of hypoxia and hypoxia-regulated proteins in cancer progression, no attempts have been made to identify hypoxia-regulated proteins using quantitative proteomics combined with MALDI-mass spectrometry imaging (MALDI-MSI). Here, we present a comprehensive hypoxic proteome study and are the first to investigate changes in situ using tumour samples. In vitro quantitative mass spectrometry analysis of the hypoxic proteome was performed on breast cancer cells using stable isotope labelling with amino acids in cell culture (SILAC). MS analyses were performed on Laser capture micro-dissected samples isolated from normoxic and hypoxic regions from tumours derived from the same cells used in vitro. MALDI-MSI was used in combination to investigate hypoxia-regulated protein localisation within tumour sections. Here, we identified more than 100 proteins, both novel and previously-reported, that were associated with hypoxia. Several proteins were localised in hypoxic regions as identified by MALDI-MSI. Visualisation and data extrapolation methods for the in vitro SILAC data were also developed, and computational mapping of MALDI-MSI data to IHC results was applied for data validation. The results and limitations of the methodologies described are discussed.