Protein mistranslation is the process by which proteins are synthesised incorrectly, resulting in polypeptides that differ from the sequence encoded by mRNA. Compared to the high-fidelity process of DNA replication, which harbours an error rate of 10-9 to 10-10, protein translation is much more error prone with an observed error rate of approximately 10-3 to 10-4. Several instances of protein mistranslation have been shown to confer fitness advantages to the organism through cellular adaptability, complexity and flexibility. For example, mistranslation creates diversity in the surface proteins of Candida albicans influencing its interactions with its host. There are three broad categories in which protein mistranslation can occur, which are amino acid misincorporation, which creates a polypeptide with an incorrect amino acid, and stop codon readthrough and frameshifting, which both typically result in a polypeptide with a different length from the intended protein. However, the degree to which these mistranslation events occur is not well understood and it is not known if there are biological factors that cause a high incidence of mistranslation events at particular sites. To address this, we are developing and applying methods to systematically detect mistranslation events in yeast mass spectrometry-based proteomics data. To detect mistranslation events in an unbiased way, “open” database searches were utilised. To ensure high quality detection of mistranslation events, filtering parameters were optimised to maximise the detection of true mistranslation events while minimising the detection of false positives. Existing datasets featuring engineered tRNA variants to induce specific types of mistranslation, carried out by the Berg lab, were analysed to establish baseline mistranslation rates and optimise filtering parameters. Putative mistranslation events have been identified, of which some mistranslation events occur at a higher frequency than others. For one engineered tRNA, an expected elevation in detection of misincorporation from arginine to serine was observed, but misincorporation of proline to glutamic acid and of valine to proline was also observed at a much higher rate than expected. This suggests that mistranslation may be a non-random process. Future work will validate these detected mistranslation events and investigate the impact of mRNA and protein sequence and structure context on mistranslation.