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The development of STAT protein-specific inhibitors has been the focus of a number of drug discovery programs. STAT activation occurs through phosphorylation at the STAT SH2 domain, resulting in dimerization, translocation to the nucleus, and transcription of proliferative genes. Due to the functional significance of the SH2 domain in mediating multiple components of the STAT signalling cascade, many libraries of inhibitors have been designed to target the SH2 domain. This has triggered the requirement for effective high-throughput screening platforms for analyzing binding by larger chemical libraries to STAT proteins. Herein, we present strategies for the development of a high-throughput thermal denaturation-based assay for identifying STAT inhibitors as well as high-yielding recombinant expression and purification of untagged STAT1, STAT3, and STAT5 proteins. This assay reports changes in the fluorescence of a labelled peptide bound to the STAT protein as a function of increasing temperature. STAT inhibitors which displace the labelled peptide elicit a change in the melt profile, which is quantitatively determined as a change in the area under the curve. This assay offers an alternative, but complimentary, high-throughput screening strategy for identifying new inhibitors of STAT proteins as well as characterizing further, the mode of inhibition by existing libraries of compounds.Thermal denaturation of STAT protein: peptide complexes can identify inhibitors.Chemical libraries (>100 compounds) can be rapidly screened against STAT proteins.The thermofluor assay was validated using previously identified STAT inhibitors.STAT1, STAT3 and STAT5 were expressed in E. coli and purified at >15 mg per liter.