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Ribosomes are the molecular machinery that catalyse all the fundamental steps involved in the translation of mRNAs into proteins. Given the complexity of this process, the efficiency of protein synthesis depends on a large number of factors among which ribosome drop-off (i.e. the premature detachment of the ribosome from the mRNA template) plays an important role. However, an in vitro quantification of the extent to which ribosome drop-off occurs is not trivial due to difficulties in obtaining the needed experimental evidence. In this work we focus on the study of ribosome drop-off in Saccharomyces cerevisiae by using 'Ribofilio', a novel software tool that relies on a high sensitive strategy to estimate the ribosome drop-off rate from ribosome profiling data. Our results show that ribosome drop-off events occur at a significant rate also when S. cerevisiae is cultured in standard conditions. In this context, we also identified a correlation between the ribosome drop-off rate and the genes length: the longer the gene, the lower the drop-off rate., (© The Author(s) 2024. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)