Fluorescence polarization was first observed by F. Weigert in 1920. He experimented with solutions of fluorescein, eosin, and other dyes at various temperatures and viscosities. Observing that polarization increased with viscosity of the solvent and the size of the dye molecule, but decreased with an increase in temperature, he deduced that polarization increased with a decrease in mobility of the emitting species. From 1925 to 1926 Francis Perrin detailed a quantitative theory for fluorescence polarization in multiple significant publications which remain relevant to this day.
Since Perrin's contribution, the technique has grown from determining binding isotherms under heavily controlled parameters, to the study of antigen-antibody, small molecule-protein, and hormone-receptor binding interactions. A fluorescence polarization immunoassay was first described and used in the 1960s. The competitive homogenous characteristic allowed for the fluorescence polarization immunoassay to be automated much easier than other immunoassay techniques such as radioimmunoassays or enzyme-linked immunoassays.
Despite originating as a method for direct interaction studies, the technique has been adopted by high-throughput screening (HTS) since the mid 1990s to help facilitate the drug discovery process by studying complex enzymatic interaction.