Abstract IL-1β is an important inflammatory cytokine of the innate immune system, released from monocytes, macrophages, and other cell types in response to a variety of pathogens and danger signals consequent to inflammasome activation. To enable direct measurement of released IL-1β from cells (no transfers or wash steps), two anti-human IL-1β antibodies were respectively labeled with complementary subunits, SmBiT and LgBiT, of the NanoLuc® Binary Technology. When the labeled antibodies bind to released IL-1β, the complementary subunits are brought into proximity, thereby reconstituting a bright luciferase that generates light when the furimazine substrate is added. Luminescence is monitored with a standard plate-reading luminometer. This novel assay effectively detected released IL-1β from differentiated human monocytic THP-1 cells and human peripheral blood mononuclear cells (PBMCs) stimulated with several TLR agonists, including endotoxin and non-endotoxin pyrogens, and could enable a simplified method for a Monocyte Activation Test for pyrogen detection. Released IL-1β can be detected directly in cell culture assay wells or in a secondary plate with transferred culture medium. Benchmarking assay performance to a standard IL-1β ELISA verified the accuracy of this more rapid, homogeneous IL-1β assay method. Of note, this IL-1β assay can be multiplexed with a homogeneous caspase-1 activity assay, providing complementary high throughput methods to identify modulators of inflammasome activation. MCC950, an NLRP3 inflammasome inhibitor, was shown to inhibit IL-1β release from treated THP-1 cells and PBMCs. Application of this homogeneous immunoassay approach to other inflammatory cytokines is currently being explored.