Ototoxicity is a main dose-limiting factor in the clinical application of aminoglycoside antibiotics. Despite longstanding research efforts， our understanding of the mechanisms underlying aminoglycoside ototoxicity remains limited. Here we report the discovery of a novel stress pathway that contributes to aminoglycoside-induced hair cell degeneration. Modifying the previously developed bioorthogonal noncanonical amino acid tagging method， we used click chemistry to study the role of protein synthesis activity in aminoglycoside-induced hair cell stress. We demonstrate that aminoglycosides inhibit protein synthesis in hair cells and activate a signaling pathway similar to ribotoxic stress response， contributing to hair cell degeneration. The ability of a particular aminoglycoside to inhibit protein synthesis and to activate the c-Jun N-terminal kinase (JNK) pathway correlated well with its ototoxic potential. Finally， we report that a Food and Drug Administration-approved drug known to inhibit ribotoxic stress response also prevents JNK activation and improves hair cell survival， opening up novel strategies to prevent and treat aminoglycoside ototoxicity.