Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated inflammatory host response to infection. Melatonin has antioxidant and anti-inflammatory activity and is currently under trial as a treatment for sepsis. The inflammasome is a cytosolic protein complex that intensifies inflammation in response to signals pathogen- and danger-associated molecular patterns, extracellular adenosine 5′-triphosphate (ATP), metabolic dysfunction, K+ efflux, and mitochondrial DNA. Once active, the inflammasome triggers caspase-1-dependent maturation of interleukin (IL)-1β, an important pro-inflammatory cytokine. Nigericin is a polyether ionophore which catalyses exchange of K+. The aim of this study was to develop an inflammasome activation model in macrophages under conditions mimicking sepsis using nigericin as a specific activator, in order to determine the effects of melatonin. In this study we describe the characterisation of the model.