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Protein misfolding is a common intracellular occurrence. Most mutations to coding sequences increase the propensity of the encoded protein to misfold. These misfolded molecules can have devastating effects on cells. Despite the importance of protein misfolding in human disease and protein evolution, there are fundamental questions that remain unanswered, such as, which mutations cause the most misfolding? These questions are difficult to answer partially because we lack high-throughput methods to compare the destabilizing effects of different mutations. Commonly used systems to assess the stability of mutant proteinsin vivooften rely upon essential proteins as sensors, but misfolded proteins can disrupt the function of the essential protein enough to kill the cell. This makes it difficult to identify and compare mutations that cause protein misfolding using these systems. Here, we present a novelin vivosystem named Intra-FCY1that we use to identify mutations that cause misfolding of a model protein [yellow fluorescent protein (YFP)] inSaccharomyces cerevisiae. The Intra-FCY1system utilizes two complementary fragments of the yeast cytosine deaminase Fcy1, a toxic protein, into which YFP is inserted. When YFP folds, the Fcy1 fragments associate together to reconstitute their function, conferring toxicity in media containing 5-fluorocytosine and hindering growth. But mutations that make YFP misfold abrogate Fcy1 toxicity, thus strains possessing misfolded YFP variants rise to high frequency in growth competition experiments. This makes such strains easier to study. The Intra-FCY1system cancels localization of the protein of interest, thus can be applied to study the relative stability of mutant versions of diverse cellular proteins. Here, we confirm this method can identify novel mutations that cause misfolding, highlighting the potential for Intra-FCY1to illuminate the relationship between protein sequence and stability.