Inflammasomes are large intracellular protein complexes that assemble in response to danger signals and function as key components of the innate immune system. Different types of inflammasomes respond to different activating stimuli and once assembled, they activate Caspase-1, triggering the secretion of IL-1β and IL-18 and the induction of pyroptosis, a lytic and inflammatory form of cell death, via cleavage of Gasdermin D. Inflammasomes are essential for host defence, but their unrestrained action contributes to a spectrum of inflammation-related illnesses, notably neurodegenerative diseases such as Alzheimer’s and Parkinson’s. The NLRP3 inflammasome responds to various damage- and pathogen-associated molecular patterns and is an extensively validated therapeutic target for neurodegenerative diseases. Here we present the optimisation and validation of a series of in vitro cell-based assays designed to support the preclinical development of novelNLRP3 inhibitors. By  combining increasingly complex and physiologically relevant human and rodent models with a comprehensive suite of readouts and validated controls, our assay cascade accelerates and de-risks the progression from concept to clinic.