Despite a rising demand for anti-obesity therapeutics, few effective pharmacological options are clinically available that target the synthesis and accumulation of body fat. Moderate inhibition of mammalian glycerol-3-phosphate acyltransferase (GPAT) with 2-(alkanesulfonamido)benzoic acids has recently been described in vitro, accompanied by promising weight loss in vivo. In silico docking studies with 2-(octanesulfonamido)benzoic acid modeled into the active site of squash GPAT revealed an unoccupied volume lined with hydrophobic residues proximal to C-4 and C-5 of the benzoic acid ring. In an effort to produce more potent GPAT inhibitors, a series of 4- and 5-substituted analogs were designed, synthesized, and evaluated for inhibitory activity. In general, compounds containing hydrophobic substituents at the 4- and 5-positions, such as biphenyl and alkylphenyl hydrocarbons, exhibited an improved inhibitory activity against GPAT in vitro. The most active compound, 4-([1,1'-biphenyl]-4-carbonyl)-2-(octanesulfonamido)benzoic acid, demonstrated an IC50 of 8.5 µM and represents the best GPAT inhibitor discovered to date. Conversely, further substitution with hydroxyl or fluoro groups, led to a 3-fold decrease in activity. These results are consistent with the presence of a hydrophobic pocket and may support the binding model as a potential tool for developing more potent inhibitors.