The biogenesis and maintenance of mitochondria relies on a sizable number of proteins. Many of these proteins are organized into complexes, which are located in the mitochondrial inner membrane. Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a method for the isolation of intact protein complexes. Although it was initially used to study mitochondrial respiratory chain enzymes, it can also be applied to other protein complexes. The use of BN-PAGE has increased exponentially over the past few years and new applications have been developed. Here we review how to set up the basic system and outline modifications that can be applied to address specific research questions. Increasing the upper mass limit of complexes that can be separated by BN-PAGE can be achieved by using agarose instead of acrylamide. BN-PAGE can also be used to study assembly of mitochondrial protein complexes. Other applications include in-gel measurements of enzyme activity by histochemical staining and preparative native electrophoresis to isolate a protein complex. Finally, new ways of identifying protein spots in Blue Native gels using mass spectrometry are briefly discussed.