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Base editing shows promise for the correction of human mutations at a higher efficiency than other repair methods and is especially attractive for mutations in large genes that are not amenable to gene augmentation therapy. Here, we demonstrate a comprehensive workflow for in vitro screening of potential therapeutic base editing targets for the USH2A gene and empirically validate the efficiency of adenine and cytosine base editor/guide combinations for correcting 35 USH2A mutations. Editing efficiency and bystander edits are compared between different target templates (plasmids versus transgenes) and assays (Next generation sequencing versus Sanger), as well as comparisons between unbiased empirical results and computational predictions. Based on these observations, practical assay recommendations are discussed. Finally, a humanized knock-in mouse model was created with the best-performing target, the nonsense mutation c.11864G>A p.(Trp3955*). Split-intein AAV9 delivery of editing reagents resulted in the restoration of USH2A protein and a correction rate of 65 ± 3% at the mutant base pair and of 52 ± 3% excluding bystander amino acid changes. This efficiency is higher than that seen in a retinal gene editing program testing in a clinical trial. These results demonstrate the effectiveness of this overall strategy to identify and test base editing reagents with the potential for human therapeutic applications.