Cyclic peptides containing unnatural amino acids can modulate Lys-48 ubiquitin chains in cells and animals. Cancer and other disease states can change the landscape of proteins post-translationally tagged with ubiquitin (Ub) chains. Molecules capable of modulating the functions of Ub chains are potential therapeutic agents, but their discovery represents a significant challenge. Recently, it was shown that de novo cyclic peptides, selected from trillion-member random libraries, are capable of binding particular Ub chains. However, these peptides were overwhelmingly proteinogenic, so the prospect of in vivo activity was uncertain. Here, we report the discovery of small, non-proteinogenic cyclic peptides, rich in non-canonical features like N -methylation, which can tightly bind Lys48-linked Ub chains. These peptides engage three Lys48-linked Ub units simultaneously, block the action of deubiquitinases and the proteasome, induce apoptosis in vitro , and attenuate tumor growth in vivo . This highlights the potential of non-proteinogenic cyclic peptide screening to rapidly find in vivo -active leads, and the targeting of ubiquitin chains as a promising anti-cancer mechanism of action.