Biosensors based on FRET have been useful in deciphering the dynamics of protein activation events in living cells at subcellular resolutions and in time scales of seconds. These new systems allow observations of dynamic processes which were not possible previously using more traditional biochemical and cell biological approaches. The image data sets obtained from these sensors require careful processing in order to represent the actual protein activation events. Here, we will cover the basic approaches useful for processing the raw image data sets into relativistic ratiometric measurements, capable of depicting relative differences in the protein activation states within a single cell. We will discuss in detail the approaches for genetically encoded, single-chain biosensor systems based on FRET, as well as those that are based on intermolecular, dual-chain design. Additionally, the same analysis can be utilized for biosensor systems using solvatochromic dyes (Nalbant, Hodgson, Kraynov, Toutchkine, & Hahn, 2004), useful for detection of endogenous protein activation states.