Antimicrobial resistance is one of the top global public health concerns that is responsible for over one million annual deaths, according to the World Health Organization. Gram-negative bacteria present a major therapeutic challenge because they possess two membranes, an inner and outer membrane separated by a periplasmic space. This makes them highly selective of molecules that enter the cell, including antibiotics. Since this membrane design acts as an impervious ‘first line of defense’ against any threat to the bacterial cell, developing inhibitors targeted at weakening this shield is a top priority. Gram-negative bacteria have become resistant to several antibiotics that target their membrane, including colistin and β-lactams, increasing the urgency of identifying alternative treatment options. This study investigates LptA, an Escherichia coli protein localized in the periplasmic space. LptA is a vital component of a transport system that facilitates the movement of lipopolysaccharide (LPS) from the cytoplasm to the outer membrane. LPS plays a critical role as a barrier against environmental stress, maintaining the integrity and structure of the outer membrane, and it is associated with antibiotic resistance. Terminating LPS transport using inhibitors of LptA would make E. coli cells more susceptible to the host’s innate immune system and antibiotics. We purified LptA to investigate strategies for inhibiting this protein. A BL21(DE3) expression strain containing a plasmid with a gene encoding the E. coli LptA was developed. This strain was used to grow bulk cultures and overexpress the protein, which was purified under native conditions using Ni-NTA affinity chromatography. We employed computer-aided drug discovery techniques to conduct a virtual screen and identified small molecules predicted to bind LptA. Preliminary binding experiments conducted using microscale thermophoresis confirmed the ability of E. coli LPS extracts to bind to the purified LptA in vitro (Kd = 208 μM). Similar experiments with a mini-library of small molecules identified from computational studies revealed three promising compounds, glecaprevir, venetoclax, and rapamycin, as potential inhibitors of LptA activity. This preliminary data opens new avenues for investigating the impact of inhibiting LptA on the susceptibility profiles E. coli cells against different antibiotics.