Mitochondria are key organelles that play a crucial role in cellular homeostasis. Dysfunction of these organelles is associated with a wide range of human diseases.Therefore, mapping the different components of mitochondria would provide invaluable information to gain further understanding of mitochondrial functions and mitochondriaassociated diseases. The mitochondrial inner membrane (MIM) contains a variety of proteins that are still unknown at their molecular level but are thought to play an essential role in several cellular processes including oxidative stress, cell death and transport of ions or metabolites. Here, we have used a new proteomics-based approach to establish a proteome of the MIM. This approach combines the use of highly purified mouse liver MIM, extraction of membrane proteins with organic acid and two-dimensional liquid chromatography coupled to mass spectrometry. This procedure allowed us to identify 182 different proteins that are involved in several biochemical processes, such as the electron transport, protein import, metabolism and ion or metabolite transport. The full range of isoelectric points, molecular masses and hydrophobicity values were represented in our list of proteins. Amongst the 182 proteins identified, 20 were unknown or had never previously been associated with the MIM. Altogether, this study demonstrates that the proteomics-based approach we have used is a powerful technique to identify new mitochondrial membrane proteins.