The present invention relates to methods, compositions, compounds and kits for detecting, measuring and modulating protein ubiquitylation via the N-end rule pathway and for identifying novel substrates, enzymes and modulators of N-end rule ubiquitylation. The present invention also relates to specific substrates of N-end rule ubiquitylation as well as activated fragments of these substrates, proteases that expose N-degrons in these substrates, ubiquitin ligases that ubiquitylate these substrates and inhibitors of the ubiquitylation of these substrates.