It is widely known that the progress of proteomics mostly depends on the development of more advanced and sensitive protein separation technologies. Immobilized metal affinity chromatography (IMAC) is a useful protein fractionation method used to enrich metal associated proteins. IMAC represents an affinity separation approach based on the interaction between proteins and metal ions immobilized on a solid support. By changing various metal ions and other experimental conditions such as pH and elution composition, IMAC can selectively isolate metal-binding protein fractions for further specific proteomic analysis. The combination of IMAC with other protein analytical technologies has been successfully applied in characterizing metalloproteome and post-translational modifications. In the future, newly developed IMAC techniques integrated with other proteomic methods will significantly contribute to the further development of proteomics.