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 abstractdeubiquitinating enzymes ( dubs ) are proteases that fulfill crucial roles in the ubiquitin ( ub ) system , by deconjugation of ub from its targets and disassembly of polyub chains . 
 the specificity of a dub towards one of the polyub chain linkages largely determines the ultimate signaling function . 
 we present a novel set of diubiquitin fret probes , comprising all seven isopeptide linkages , for the absolute quantification of chain cleavage specificity of dubs by means of michaelis  menten kinetics . 
 each probe is equipped with a fret pair consisting of rhodamine110 and tetramethylrhodamine to allow the fully synthetic preparation of the probes by spps and ncl . 
 our synthetic strategy includes the introduction of n , nbocprotected 5carboxyrhodamine as a convenient building block in peptide chemistry . 
 we demonstrate the value of our probes by quantifying the linkage specificities of a panel of nine dubs in a highthroughput manner . 
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