Förster resonance energy transfer-based analytical techniques represent a unique tool for bioanalysis because they allow one to detect protein-protein interactions and conformational changes of biomolecules at the nanometer scale, both "in vitro" and "in vivo" in cells, tissues and organisms. These techniques are applied in diverse fields, from the detection and quantification of ligands able to bind to proteins or receptors to the development of RET-based whole-cell biosensors, microscope imaging techniques and "in vivo" whole-body imaging for the monitoring of physiological and pathological processes. However, their quantitative performances need further improvements and, even though RET measurement principles and procedures have been continuously improved, in some cases only qualitative or semiquantitative information can be obtained. In this review we report recent applications of RET-based analytical techniques and discuss their advantages and limitations.