First, a cell lysate is generated by glass beads beating, pressure homogenisation or chemical or physical lysis methods that do not denature the protein(s) of interest. (Optionally for targeted analysis) a protein of interest is purified out of this lysate by affinity methods based on intrinsically disordered tags  or other suitable purification strategies, often involving several orthogonal chromatographic steps.
This (total or purified) protein solution is aliquoted into several tubes of a PCR strip. All aliquots are exposed in parallel in a thermal gradient PCR cycler to different maximal temperatures in presence of the thermostable protease thermolysin (see figure). 
Automated temperature control is achieved in a thermal gradient cycler (commonly used for PCRs). Reaction products can be separated by SDS-PAGE or western blot.  The protease thermolysin can be fully inactivated by EDTA. This feature of thermolysin makes FASTpp compatible with subsequent trypsin digestion e.g. for mass spectrometry.