Updated project metadata. Multi-drug resistant Gram-negative bacteria (GNB) are major contributors to the anti-microbial resistance (AMR) burden in humans and animals. AMR mechanisms are primarily mediated by proteoforms and, therefore, proteomic analyses of GNB offers a significant advantage in understanding the mechanisms of AMR. A large portion of these mechanisms are mediated by membrane proteins, however, they are often difficult to extract due to their hydrophobic nature and complex interactions with other components of the cell membrane. To extract the greatest number of proteoforms, an efficient ho-mogenisation protocol is required to effectively disrupt the rigid cell wall and membrane. Using Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa we systematically compared the extraction efficiency of bead-beating with flash frozen and lyophilized cell pellets. We demonstrate that lyophilization prior to ho-mogenisation by bead-beating increases the detection of hydrophobic, membrane pro-teins. We detected numerous unique membrane proteins in each bacterial isolate, in-cluding ABC transporters and proteins involved in lipopolysaccharide synthesis, when lyophilizing prior to bead-beating compared to only flash freezing prior. As membrane proteins play a central role in AMR resistance mechanisms, this improvement in their isolation and identification will aid in understanding the resistance and molecular mechanisms associated with multi-drug resistant GNB.