This paper describes a method that allowed the measurement of target engagement by drugs in cells and tissue samples. The assay was based on the principle that ligands induce thermal stabilization of their target that confers a 'fingerprint' of drug–target engagement. In whole cells, this assay could measure levels of target engagement of several types of anticancer drugs (such as antifolates, cyclin-dependent kinase inhibitors and vemurafenib). This assay could also be used in ex vivo tissue, as demonstrated by its ability to monitor target engagement of an anti-angiogenic compound in mouse tissue.