Evolving lineages face a constant intracellular threat: most new coding sequence mutations destabilize the folding of the encoded protein. Misfolded proteins form insoluble aggregates and are hypothesized to be intrinsically cytotoxic. Here, we experimentally isolate a fitness cost caused by toxicity of misfolded proteins. We exclude other costs of protein misfolding, such as loss of functional protein or attenuation of growth-limiting protein synthesis resour- ces, by comparing growth rates of budding yeast expressing folded or misfolded variants of a gratuitous protein, YFP, at equal levels. We quantify a fitness cost that increases with misfolded protein abundance, up to as much as a 3.2% growth rate reduction when misfolded YFP represents less than 0.1% of total cellular protein. Comparable experiments on variants of the yeast gene orotidine- 5′-phosphate decarboxylase (URA3) produce similar results. Quan- titative proteomic measurements reveal that, within the cell, mis- folded YFP induces coordinated synthesis of interacting cytosolic chaperone proteins in the absence of a wider stress response, pro- viding evidence for an evolved modular response to misfolded pro- teins in the cytosol. These results underscore the distinct and evolutionarily relevant molecular threat of protein misfolding, in- dependent of protein function. Assuming that most misfolded pro- teins impose similar costs, yeast cells express almost all proteins at steady-state levels sufficient to expose their encoding genes to selection against misfolding, lending credibility to the recent sug- gestion that such selection imposes a global constraint on molec- ular evolution.