Displacement elution (DC) is an elution mode in liquid chromatography (LC) beside isocratic- and gradient chromatography (GC). In the past three decades Horvath, Cramer as well as Mant and Hodges published several convincing studies about the power of DC for the separation of peptides. However, DC is far from being popular. We applied DC successfully for preparative separations in the 1990´s already. In this study we followed the question if even complex mixtures of tryptic peptides can be separated with DC and we compared the performance of DC with GC. We digested protein extracts from cultured cells with trypsin and separated aliquots of the tryptic peptides (5 µg total amount per sample) via DC and via GC with the same chromatographic system comprising a cation exchange (SCX) as first column followed by reversed-phase (RP) capillary column coupled in an on-line two-dimensional nano-liquid-chromatography system coupled to a tandem mass spectrometer (2D-LC-MSMS). The peptides eluting from the RP column were desorbed and ionized by an electrospray-ionization source to an MSMS. LC-MSMS data were processed and proteins identified and quantified with algorithms uploaded from MaxQuant. The resolution of the SCX separation in the DC-mode was significantly higher than that of the GC-mode with respect to the number of identified peptides. Especially those tryptic peptides, being doubly protonated (the majority of tryptic peptides) in the formic acid sample application buffer of the SCX eluent system, on the SCX column in the DC mode were separated with a much higher resolution than in the GC mode. In addition the reproducibility of the DC mode was higher. In conclusion our comprehensive study and comparison of the DC and GC mode demonstrate that DC is very powerful even for the separation of extremely complex peptide mixtures, which we usually have to deal with in proteomics.