In this chapter, we describe the use of immunohistochemical methods to detect hypoxia in tumour tissue sections, utilising antibodies specific for endogenous proteins hypoxia inducible factor 1 alpha (Hif1α) and glucose transporter 1 (Glut-1) and the exogenous compound pimonidazole (Pim). Immunohistochemistry is routinely used both diagnostically and in research to label and identify specific cellular proteins of interest. The methods described here enable staining of hypoxic cells and tissue in formalin-fixed paraffin-embedded (FFPE) tumour tissue sections that can then be visualised either using chromogenic or fluorescence detection. All three methods can be used on human, xenograft, or mouse tumour tissue.