Morphogens are signaling molecules that provide positional information to cells during development. They must move through embryonic tissues in order to coordinate patterning. The rate of a morphogen's movement through a tissue-its effective diffusivity-affects the morphogen's distribution and therefore influences patterning. Fluorescence recovery after photobleaching (FRAP) is a powerful method to measure the effective diffusion of molecules through cells and tissues, and has been successfully employed to examine morphogen mobility and gain important insights into embryogenesis. Here, we provide detailed protocols for FRAP assays in vitro and in living zebrafish embryos, and we explain how to analyze FRAP data using the open-source software PyFRAP to determine effective diffusion coefficients.