As scavengers, spotted hyenas (Crocuta crocuta) are exposed to a wide array of pathogens but exhibit low mortality rates due to infectious disease. This suggests that this species exhibits a unique and robust immune response to pathogens. However, few tools exist to measure cell-mediated immunity (CMI) in hyenas and we aimed to develop a gene expression assay to quantify antigen-specific responses. Whole blood from five Mycobacterium bovissensitised hyenas was incubated in Nil and TB antigen tubes of the QuantiFERON^®-TB Gold (QFT) system. Using qPCR, the relative expression stability of the reference genes ACTB, GAPDH, YWHAZ and TBP in these samples was determined as well as the mean fold change in the expression of IFNG, CXCL8, CXCL9, CXCL10 and CXCL11 in M. bovis-antigen stimulated blood. The expression of YWHAZ and TBP showed greatest stability, and YWHAZ was selected as a reference for further analysis. The expression of CXCL9 and CXCL11 showed greatest upregulation in antigen-stimulated blood and the assay results for these genes were strongly correlated. The measurement of antigen-induced CXCL9 and CXCL11 expression, relative to that of YWHAZ, can be used to measure CMI responses to infectious diseases in spotted hyenas.