We describe an antibacterial assay based on bioluminescence of two Gram negative bacteria, Photorhabdus luminescens and transformed Escherichia coli, which can be used as a real-time measurement of antibacterial activity in insect haemolymph. This method is based on the production of the bioluminescence signal depending on the viability of bacterial cells. We observed a significant rapid dose-dependent decrease in bioluminescence using both bacterial species, and Bombyx mori or Galleria mellonella haemolymph, which was confirmed by the decrease in bacterial viability determined by plating. The humoral origin of the antibacterial activity observed in whole haemolymph was confirmed for haemolymph plasma without haemocytes. Antibacterial activity directed against Gram negative bacteria was recorded in unaffected insect larvae as well as after septic injury; increased antibacterial activity of haemolymph was detected in the latter case confirming the inducibility of antimicrobial agents. We think it is likely that this method could be widely used for determining antibacterial activity in insects and other invertebrates.