Super resolution techniques are becoming increasingly popular but are often incompatible with live imaging. Moreover, their combination with the implementation of advanced quantitative is challenging. In this workshop, we aim at providing a straightforward protocol to estimate FRET in the context of super-resolution images obtained with SRRF (Super Resolution Radial Fluctuations). As a biological paradigm, we will work with a well-established FRET biosensor detecting the activation of the AURKA kinase. The activation of the sensor will be followed in a particular subcellular compartment of interest such as mitochondria, but the protocol can be applied to other subcellular compartments or other biosensors of interest. Users attending the workshop will be able to detect the activation of AURKA by measuring FRET of 3 constructs: an active biosensor, an inactive kinase-dead variant or a donor-only “FRETless” counterpart. FRET will be measured in the ratiometric mode. We are confident that this workshop will provide the attendees with the conceptual and practical tools to readily adapt FRET measurements on the widefield or spinning-disk setups available in their closest facility.