Protein misfolding is a common intracellular occurrence that has devastating effects. Misfolded proteins are cytotoxic and their accumulation within the cell can impose a fitness cost that usually manifests as a growth rate reduction. Therefore, isolating and measuring their effects on fitness are integral to understanding the significance of misfolded protein toxicity. There are also basic questions about protein misfolding that remain unanswered, such as, what mutations cause protein misfolding, how does the harm that misfolded proteins cause scale with their abundance within the cell, and why misfolded proteins are toxic. Here, we present a high-throughput system to accurately measure the fitness effects of thousands of misfolded protein variants and to understand the mechanistic basis of their toxicity. Through massively parallel genome editing, competitive growth assays, and next generation sequencing, we study over 2,000 barcoded unique misfolding mutations to either yellow fluorescent protein (YFP) or superoxide dismutase 1 (Sod1) in Saccharomyces cerevisiae. We express these proteins such that observed fitness costs are likely due to the gain of cytotoxic misfolded proteins, rather than the loss of any beneficial protein function. Through this system, we measure the fitness defect associated with each mutation. We also devise a novel technique that we name Intra-FCY1 to estimate how much misfolding is caused by each of our thousands of mutations. Unlike western blots, this orders-of-magnitude higher-throughput technique uses relative growth rates to measure the amount of misfolded protein within each barcoded yeast strain. Our comprehensive screen of mutations in these two proteins reveals which mutations cause the most severe misfolding, and shows how the fitness effects of protein misfolding scale as misfolding becomes more severe. Our Intra-FCY1 system, surveying 2,000 distinct mutants per protein, represents a notable advancement in investigating the effects of misfolded proteins on cells. This advancement will inform questions in the field of evolutionary biology about the distribution of fitness effects (DFE) for new mutations, since the majority of mutations in protein-coding sequences tend to increase the chance of the resulting protein being misfolded.