Rationale and Objectives: Dithiothreitol (DTT) is commonly used to liquefy induced sputum samples prior to assessment of cytology, but causes reduction of disulphide bonds and denaturation of proteins. The objective of this study was to process sputum supernatants containing DTT to enable quantification of cytokines and chemokines. Methods: A standard solution of 22 pooled chemokines and cytokines was incubated with DTT at the concentrations used during sputum liquefaction and then dialysed in 20 different denaturant and redox conditions. Results: Following incubation of the standard solution with DTT there was loss of detectable protein mediators on immunoassay, but optimised dialysis permitted recovery of chemokines to 96±4% and cytokines to 91±6%. Optimised dialysis of DTT supernatants from asthmatics of a range of severities (n=35) was performed in the presence of a cocktail of protease inhibitors and demonstrated significantly elevated levels of the chemokine CXCL10 (IP-10), CXCL8 (IL-8) and CCL3 (MIP-1); with lower but significantly elevated levels of CCL2 (MCP-1), CCL11 (eotaxin) and CCL5 (RANTES) in severe asthma. In sputum from severe asthmatics there were also significantly elevated levels of IL-4, IL-5, IL-13, TNF-, IL-6, GM-CSF and IL-12 (p40). Conclusions: The technique of optimised dialysis and protease inhibition of sputum DTT supernatants aids the detection of chemokines and cytokines. The detection of elevated levels of particular sputum chemokines and cytokines in individual patients may provide a rationale for specific therapies.