Immune regulation is key in health and disease and is sustained via a balance of multiple cellular and molecular mechanisms. These mechanisms can be roughly grouped into those promoting inflammation and those that keep inflammation in check while promoting tissue healing. NOD-like receptors are a large protein family that include many key pro- and anti-inflammatory signalling molecules. The NLRP subfamily of NOD-like receptors includes the well-characterised inflammasome scaffolds NLRP3 and NLRP6, as well as a number of poorly characterised members whose functions in immune regulation remain controversial. NLRP7 and NLRP12 are proposed to form inflammasomes, with NLRP12 being a favourite inflammasome-nucleating candidate, due to its association with a severe genetic inflammatory disorder, FCAS2, that closely resembles the FCAS disorder caused by gain-of-function NLRP3 mutations. Here, we report the first unbiased screen for inflammasome function within the NLRP subfamily, taking advantage of a novel unbiased and quantitative approach, Time-of-Flight Inflammasome Evaluation (TOFIE), to identify bona fide inflammasome nucleators within the NLRP subfamily. We report that NLRP3 and NLRP6 initiate ASC polymerisation into a “speck” in our system, confirming their known biology. We show that, surprisingly, NLRP12 cannot nucleate ASC specks, due to a functional deficiency in its NACHT domain and the very strong inhibitory potential of its LRR domain. In the light of its close resemblance to NLRP3, we hypothesised that NLRP12 may instead inhibit NLRP3 function, which was indeed the case. These findings suggest that the previously reported patient-associated NLRP12 mutations may result in abrogated inhibitory function, rather than enhanced NLRP12 inflammasome function. Given that both NLRP3 and NLRP12 are associated with severe auto-inflammatory diseases, our study provides important insight into inter-regulated NLRP3 and NLRP12 biology that suggests new therapeutic approaches for auto-inflammatory disease management. ​