Cellular proteins are degraded by the ubiquitin-proteasome system (UPS) in a precise and timely fashion. Such precision is conferred by the high substrate specificity of ubiquitin ligases. Identification of substrates of ubiquitin ligases is crucial not only to unravel the molecular mechanisms by which the UPS controls protein degradation but also for drug discovery purposes because many established UPS substrates are implicated in disease. We developed a combined bioinformatics and affinity purification– mass spectrometry (AP-MS) workflow for the system-wide identification of substrates of SCF bTrCP , a member of the SCF family of ubiquitin ligases. These ubiquitin ligases are characterized by a multi-subunit architecture typically consisting of the invariable subunits Rbx1, Cul1, and Skp1, and one of 69 F-box proteins. The F-box protein of this member of the family is bTrCP. SCF bTrCP binds, through the WD40 repeats of bTrCP, to the DpSGXX(X)pS diphosphorylated motif in its substrates. We recovered 27 pre-viously reported SCF bTrCP substrates, of which 22 were verified by two independent statistical proto-cols, thereby confirming the reliability of this approach. In addition to known substrates, we identified 221 proteins that contained the DpSGXX(X)pS motif and also interacted specifically with the WD40 repeats of bTrCP. Thus, with SCF bTrCP , as the example, we showed that integration of structural infor-mation, AP-MS, and degron motif mining constitutes an effective method to screen for substrates of ubiquitin ligases. INTRODUCTION