Hypoxia is a ubiquitous feature of many tumours contributing to disease progression and treatment resistance, and therefore represents a well-validated physiological target for cancer therapy. Several hypoxia-activated prodrugs have been developed at the University of Auckland. However, further clinical development requires identifying tumours that are hypoxic, express prodrug-activating reductases, and are intrinsically sensitive to the activated drug. Profiling of gene expression typically uses RNA-based methods, but most phenotypes are more directly linked to the protein expression. Here we employed a targeted proteomics strategy to develop an assay for candidate prodrug-activating reductases and endogenous markers of hypoxia with a group of housekeeping proteins for nomalisation. By employing stable isotope-labelled peptide standards we performed absolute quantitation of the proteotypic peptides in head and neck squamous cell carcinomas (HNSCC) cell lines and xenografts. The results showed variable reductase expression across a HNSCC panel and corresponding mouse xenografts, with CYB5R3 and NQO1 exhibiting high expression, and POR, AKR1C3, TXNRD1 and FDXR in an intermediate range. T This is consistent with the observation that CYB5R3, NQO1 and TXNRD1 transcripts are amongst the most abundant mRNAs encoding prodrug-activating reductases across two HNSCC clinical cohorts. Seven detectable endogenous hypoxia markers, which have been clinically validated at the mRNA level as hypoxia classifiers in HNSCC, showed protein upregulation under chronic hypoxia in cell culture. In conclusion, the multiplexed assay is suitable for assessing absolute levels of reductase expression in cell lines and tumours, and there is promising potential to integrate additional biomarkers for hypoxia and genes that determine intrinsic sensitivity in the future to build powerful predictive biomarkers assay capacity to support drug development and clinical application.