ABSTRACT Signal transduction pathways are intricately fine-tuned to accomplish diverse biological processes. An example is the conserved Ras/mitogen-activated-protein-kinase (MAPK) pathway, which exhibits context-dependent signaling output dynamics and regulation. Here, by altering codon usage as a novel platform to control signaling output, we screened the Drosophila genome for modifiers specific to either weak or strong Ras-driven eye phenotypes. We mapped the underlying gene from one modifier to the ribosomal gene RpS21 . RpS21 preferentially influences weak Ras/MAPK signaling outputs, and negatively regulates Ras/MAPK in multiple cell/tissue and signaling settings. In turn, MAPK signaling may regulate its own negative feedback by promoting RpS21 expression. These data show that codon usage manipulation can identify output-specific signaling regulators, and identify RpS21 as an in vivo Ras/MAPK phenotypic regulator.