The inner nuclear membrane (INM) is a functional sub compartment of the ER, though is quite dissimilar from it. With a very different protein content from the general ER, this compartment’s complete proteome has not been determined. This specificity is largely based on the restrictions enforced by the nuclear pore complex (NPC) for nuclear import of membrane proteins. The best characterised model for this process (diffusion retention model) suggests that the NPC imposes a diffusion barrier to proteins with extralumenal domains larger than 60 90 kDa; and also that the proteins which are indeed trafficked across the pore can then be retained in the INM due to interactions with lamins and/or chromatin, or proteins associated with either of the two. Even though in the last couple decades many advances have been made surrounding this sub compartment, each had a set of restrictions, the biggest one being the reliance on membrane fractionation procedures and detergent extraction based methods to answer the question. In an attempt to overcome these limitations, this project was designed with the aim of establishing a biochemical assay capable of determining the protein composition of the INM of mammalian cells in situ. For that purpose, we chose to employ an engineered ascorbate peroxidase that very efficiently biotinylates proximal proteins named APEX2. By covalently modifying the proteins of this compartment, we simply need to create a total cell extract, from which we can separate these biotinylated proteins in a pull down and analyze them using tandem mass spectrometry. To do so, cell lines expressing APEX2 fused to a nuclear localization signal (NLS) or an INM resident protein, Lamin B receptor (LBR), were generated using different strategies as to control their expression levels, in an attempt to attain a nuclear and nuclear rim specific localization, respectively. Furthermore, the protocol for inducing APEX2’s biotinylation and pull down for mass spectrometry analysis was also tested. Further optimisation is still needed, though we believe this study sets some guidelines for additional studies involving the INM and its intriguing characteristics. Hence, it can be the starting point for more research focusing on other questions surrounding this sub compartment, namely the turnover rates of these proteins and what influences them, the quality control mechanisms employed to preserve the INM’s very particular proteome or the mechanisms of nuclear envelope (NE) reformation and how this INM identity is maintained even in cells that divide employing an open mitosis.