Fluorescence recovery after photobleaching (FRAP) is a fluorescence microscopy technique that has attracted a lot of interest in pharmaceutical research during the last decades. The main purpose of FRAP is to measure diffusion on a micrometer scale in a non-invasive and highly specific way, making it capable of measurements in complicated biomaterials, even in vivo. This has proven to be very useful in the investigation of drug diffusion inside different tissues of the body and in materials for controlled drug delivery. FRAP has even found applications for the improvement of several medical therapies and in the field of diagnostics. In this review, an overview is given of the different applications of FRAP in pharmaceutical research, together with essential guidelines on how to perform and analyse FRAP experiments.