Detection of Gram-negative bacterial lipid A by the extracellular sensor, MD-2/TLR4 or the intracellular inflammasome sensors, CASP4 and CASP5, induces robust inflammatory responses. The chemical structure of lipid A, specifically the phosphorylation and acylation state, varies across and within bacterial species, potentially allowing pathogens to evade or suppress host immunity. Currently, it is not clear how distinct alterations in the phosphorylation or acylation state of lipid A affect both human TLR4 and CASP4/5 activation. Using a panel of engineered lipooligosaccharides (LOS) derived from Yersinia pestis with defined lipid A structures that vary in their acylation or phosphorylation state, we identified that differences in phosphorylation state did not affect TLR4 or CASP4/5 activation. However, the acylation state differentially impacted TLR4 and CASP4/5 activation. Specifically, all of the examined tetra-, penta-, and hexa-acylated LOS variants activated CASP4/5-dependent responses, whereas TLR4 responded to penta- and hexa-acylated LOS but did not respond to tetra-acylated LOS or pentaacylated LOS lacking the secondary acyl chain at the 3’ position. As expected, lipid A alone was sufficient for TLR4 activation; however, human macrophages required both lipid A and the core oligosaccharide to mount a robust CASP4/5 inflammasome response. Our findings show that human TLR4 and CASP4/5 detect both shared and non-overlapping LOS/lipid A structures, which enables the innate immune system to recognize a wider range of bacterial LOS/lipid A, thereby constraining the ability of pathogens to evade innate immune detection.