waaP is present in the lipopolysaccharide (LPS) core gene clusters of a wide range of gram-negative bacteria, and is an essential gene in Pseudomonas aeruginosa. The WaaP protein is a sugar kinase that adds phosphate to heptose I in the core oligosaccharide. This study describes the standardization and utility of a chemiluminescence-based enzyme-linked immunosorbent assay for the detection of WaaP kinase activity. Important features of the assay include high sensitivity, the preparation of dephosphorylated LPS as a substrate, and the use of monoclonal antibody 7-4 that specifically recognizes phosphate substituents in the LPS core.