During oxidative stress, K63-linked polyubiquitin chainsmodify a variety of proteins including ribosomes. Knowledge of the precise sites of K63 ubiquitinis key to understand its function during the response to stress. To identify the sites of K63 ubiquitin, we developed a new massspectrometry based method that quantified >1100 K63 ubiquitination sites in yeast that responded to oxidative stress induced by H2O2. We determined that under stress, K63 ubiquitin-modified proteins were involved in several cellular functions including ion transport, protein trafficking, and translation. The most abundant ubiquitinsites localized to the head of the 40S subunit of the ribosome, modified assembled polysomes, and affected the binding of translation factors. The results suggested a new pathway of post-initiation control of translation during oxidative stress and illustrated the importance of high-resolution mapping of noncanonical ubiquitination events.