Fractionation of complex samples at the cellular, subcellular, protein, or peptide level is an indispensable strategy to improve the sensitivity in mass spectrometry-based proteomic profiling. This study revisits, evaluates, and compares the most common gel-based protein separation techniques i.e. 1D SDS-PAGE, 1D preparative SDS-PAGE, IEF-IPG, and 2D-PAGE in their performance as fractionation approaches in nano LC-ESI-MS/MS analysis of a mixture of protein standards and mitochondrial extracts isolated from rat liver. This work demonstrates that all the above techniques provide complementary protein identification results, but 1D SDS-PAGE and IEF-IPG had the highest number of identifications. The IEF-IPG technique resulted in the highest average number of detected peptides per protein. The 2D-PAGE was evaluated as a protein fractionation approach. This work shows that the recovery of proteins and resulting proteolytic digests is highly dependent on the total volume of the gel matrix. The performed comparison of the fractionation techniques demonstrates the potential of a combination of orthogonal 1D SDS-PAGE and IEF-IPG for the improved sensitivity of profiling without significant decrease in throughput.