The C-terminal of proteins regulates their biological functions. Small open reading frame-encoded peptides (SEPs) are a novel class of gene expression products that participate in various biological activities. The C-termini of SEPs have also been found to affect their function, but the polymorphism of SEPs' C-terminal has not yet been systematically elucidated. Here, we utilized C-terminal proteomics technology to systematically analyze the C-termini of SEPs in human tumor cell lines. We combined two SEPs enrichment methods (ACN and heat precipitation) and two C-terminal peptides enrichment methods (MOLEX and CTAILS) to identified the C-terminal peptides of SEPs. Each experiment was performed with triplicate.