Characterization of mature protein N-termini by large scale proteomics is challenging. This is especially true for proteins undergoing cleavage of transit peptides when they are targeted to specific organelles, such as mitochondria or chloroplast. Protein neo-N-termini can be located up to 100-150 amino acids downstream from the initiator methionine and are not easily predictable. Although some bioinformatics tools are available, they usually require extensive manual validation to identify the exact N-terminal position. The situation becomes even more complex when post-translational modifications take place at the neo-N-terminus. Although N-terminal acetylation occurs mostly in the cytosol, it is also observed in some organelles such as chloroplast. To date, no bioinformatics tool is available to define mature protein starting positions, the associated N-terminus acetylation status and/or yield for each proteoform. In this context, we have developed the EnCOUNTer tool (i) to score all characterized peptides using discriminating parameters to identify bona fide mature protein N-termini and (ii) to determine the N-terminus acetylation yield of the most reliable ones.