Fluorescence techniques provide extraordinary high levels of sensitivity, specificity and selectivity and they are well-suited for high-throughput screening and imaging of specific proteins and drugs in biological samples. The most effective approach to quantify protein complexes within a sample is to use fluorescence anisotropy (FA) to quantify the formation of complexes between a FA-sensor and its target. FA has the distinct advantage over FRET in that it simply measures the increase in molecular volume of the bound FA sensor and using a single fluorophore. Currently, most FA-probes are prepared via laborious and specific chemical synthesis, reducing their appeal for high-throughput and in vivo screening of specific drugs or proteins. We will advance FA-based analyses of specific proteins for in vitro and in vivo systems through the introduction and optimization of a completely new class of genetically-encoded FA-sensor. The three sensors detailed in this proposal are truncated forms of: (i), a non-switchable mutant of Lov2 (fLov2), (ii), the yellow fluorescent protein from Vibrio fischeri (Y1); (iii), the lumazine binding protein (LUMP) from Photobacterium Leioghnati. The fluorescence properties of these flavoproteins proteins are similar to GFP, YFP and CFP respectively, although they only have 40%~67% of the mass, and exhibit far longer fluorescence lifetimes than GFP, a key property in the design of an FA-sensor for large proteins. The studies detailed are innovative on several counts and include the introduction of the smallest genetically-encoded fluorescent proteins for intracellular imaging of fused proteins, and the first encoded probes specifically designed for FA-based detection and imaging of specific protein targets in living cells. The research also identifies a promising approach for FA-based proteome-wide analysis of protein or drug interactions in vitro and in living bacteria and yeast.