Treatment of Gram-negative bacterial infections is complicated by innate and acquired drug resistance resulting in a limited number of effective antibiotics. Several Gram-negative bacteria, for which current therapies are ineffective, have recently been identified as potential bioterror agents. These findings highlight the need for new antibiotics, specifically antibiotics that act on new drug targets to circumvent drug resistance. Potential targets in Gram-negative bacteria include enzymes involved in the biosynthesis of lipopolysaccharides (LPS) that form outer membranes of these organisms. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the committed step in the biosynthesis of the lipid A portion of LPS. Therefore, inhibitors of this enzyme have the potential to serve as antibiotics, and efforts toward the development of LpxC inhibitors are currently underway. Here we describe methods for assaying LpxC inhibitors, including methods for measuring deacetylase activity and binding affinity for LpxC, which will be useful for the development of LpxC inhibitors.