Tryptic digestion of biotinylated Lys-C peptides followed by affinity chromatography allows the selective isolation of lysine-free tryptic peptides delimited by arginine residues (RRnK peptides). In silico analysis revealed that RRnK peptides represent 87% of the whole proteomes and their specific isolation simplifies the complex peptide mixture (5 peptides per protein). The good recoveries and high selectivity obtained in the isolation of RRnK peptides anticipate the applicability of this method in 2DE-free quantitative proteome analyses.