In addition to the catalytically active kinase domain, most kinases feature regulatory domains that govern their activity. Modulating and interfering with these interdomain interactions presents a major opportunity for understanding biological systems and developing novel therapeutics. Therefore, small molecule inhibitors that target these interactions through an allosteric mode of action have high intrinsic selectivity, as these interactions are often unique to a single kinase or kinase family. Here we report the development of iFLiK (interface-Fluorescent Labels in Kinases), a fluorescence-based assay that can monitor such interdomain interactions. Using iFLiK, we have demonstrated selective detection of allosteric Akt inhibitors that induce an inactive closed conformation unique to Akt. This methodology easily distinguished small molecule allosteric inhibitors from classic ATP-competitive inhibitors. Screening an in-house compound library with iFLiK, we were able to identify novel compounds with a scaffold that has not been previously described for allosteric Akt inhibitors.