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Previous publications on stapled peptide inhibitors against Mdm2/Mdm4-p53 interactions have established that this new class of drugs have the potential to be easily optimised to attain high binding affinity and specificity, but the mechanisms controlling their cellular uptake and target engagement remain elusive and controversial. To aid in understanding the rules of peptide and staple design and to enable rapid optimisation, we employed the newly-developed cellular thermal shift assay (CETSA). CETSA was able to validate stapled peptide binding to Mdm2 and Mdm4 and the method was also used to determine the extent of cellular uptake, cellular availability and intracellular binding of the endogenous target proteins in its native environment. Our data suggest that while the stapled peptides engage their targets intracellularly, more work is needed to improve their cellular entry and target engagement efficiency in …