The direct measurement of drug-protein interactions in living cells is a major challenge in drug discovery research. Using the cellular thermal shift assay or CETSA such measurements can be achieved, in principle, in any cell samples and in microtiter-plate format. This chapter starts with an overview of CETSA and then continues to provide thorough guidance in the development, optimization and application of a microplate-based protocol using AlphaScreen® as the detection format. Significant parts of the experimental descriptions are applicable also to other detection modalities. Each step in the assay development and validation process is exemplified by real case data from the development and validation of a fully screen-compatible live-cell assay for thymidylate synthase (TS; encoded by TYMS). When possible, the descriptions are kept general such that it allows for translation to other target proteins and efforts are made to point out crucial steps and considerations in the experimental design. At the end of the chapter there is a section devoted to insights from our screen adaptation experiences, troubleshooting and a discussion on tentative applications of microplate-based CETSA.