Gram-negative bacteria (GNB) are major drivers of antimicrobial resistance (AMR) in humans and animals. AMR mechanisms are primarily mediated by proteoforms, therefore, proteomic analyses of GNB offers a significant advantage in understanding the mechanisms of AMR. The power of such proteomic analyses are dependent on efficient, robust sample preparation. Common extraction protocols include: (1) cell lysis and homogenization, (2) protein extraction and solubilization, and (3) the removal of unwanted analytes or contaminants (i.e. lipids, nucleic acids, salts etc.) that may interfere downstream analyses. While GNB are relatively easy to lyse relative to other microbes (e.g Gram-positive or sporulated organisms), an efficient homogenization protocol is still required to effectively disrupt the rigid cell wall and membrane to extract proteins. Previously, it has been demonstrated that bead-beating results in improved cell lysis and extraction yields. However, the efficiency of bead-beating is dependent on various factors including bead size, frequency and speed, and cell characteristics. Using K. pneumoniae as a model organism, we systematically compare the homogenization efficiency of bead-beating with flash frozen and lyophilized cell pellets for GNB proteomic analyses. In this study, we demonstrate that lyophilization prior to homogenisation increases protein extraction yield, and enriches for hydrophobic proteins and membrane proteins, including multidrug efflux pumps and ABC transporter proteins. We detected 84 unique membrane proteins when lyophilizing prior to homogenisation compared to 8 membrane proteins detected when only flash freezing prior to homogenisation. Membrane proteins play a central role in AMR resistance mechanisms, therefore improving the capacity to isolate and identify them can further aid in understanding the resistance mechanisms and molecular mechanisms associated with multidrug-resistant GNB.