Introduction: Proteomic biomarkers may be useful for monitoring therapeutic response and to triage cancer patients to best therapy.

Objectives: In this review, we highlight the importance of specimen acquisition, preparation and analysis in radiotherapy proteomic studies. We also discuss practical approaches for the design and execution of clinical proteomic studies using our recent experience based on specimens accrued during prostate cancer radiation therapy.

Discussion and conclusions: Numerous proteomic methods are being employed, including high-throughput mass spectrometry and immunoassays, and using solid tissues, blood and urine for analysis. Given the potential complexity of cytokine and other protein responses, there is a need to assess proteomic signatures within serial samples as longitudinal studies during a course of fractionated radiotherapy (RT).