A review focusing on the development of multidimensional chromatography coupled to tandem mass spectrometry (MS)for shotgun proteomics analysis is presented. Proteomics analyses are frequently performed to obtain information ranging from simple protein identification to more complex post-translational modification studies and have extended to dynamic studies in response to various stimuli. The coupling of different chromatographic techniques like strong cation exchange/reversed phase liquid chromatography separations with the addition of affinity chromatography has granted a view into a realm previously unknown. The human serum proteome is likely to span >10 orders of magnitude for the dynamic range. Highly abundant proteins such as albumin and transferrin must be removed from the serum through affinity methods and fractionation techniques. Another way to increase the number of theoretical plates in a chromatography analysis is to have smaller particle size which then requires higher pressures for analysis. Ultrahigh-pressure reversed phase liquid chromatography has become active in this area. The implementation of UHPLC in shotgun proteomics system is now possible, but further research is needed in small particle and high-pressure resistant strong cation exchange to fully integrate such application. Moreover, advances in mass spectrometry such as coupling of linear ion trap (LTQ) to Fourier transform (FT-MS) has improved analyses of prostate cancer and of histone phosphorylation. The implementation of all these technological advances and others into shotgun proteomics will continue enabling biological discoveries.