Updated FTP location. We have developed a one-step method to isolate protein C-terminal peptides from V8-digested proteins by metal oxide-based ligand-exchange (MOLEX) chromatography. V8 protease cleaves the C-terminal side of Asp and Glu, and the resulting digested peptide has two carboxy groups at the C-terminus, whereas the protein C-terminal peptide has only one α-carboxy group. In MOLEX chromatography, a stable chelate is formed between dicarboxylates and metal atoms, so that the non-terminal (i.e., internal) peptide is retained, but the protein C-terminal peptide flows through the MOLEX column. By optimizing the conditions of V8 protease digestion and MOLEX chromatographic isolation, a total of 1619 protein C-termini from 30 μg of peptides derived from human HeLa cells could be identified in 10 min of the isolation step. Furthermore, using 200 µg of peptide, we identified 2202 protein C-termini, which was the largest data set with the least amount of sample in protein C-terminomics. These results indicated that this method is unprecedentedly simple and sensitive for protein C-terminal peptide enrichment.