Inflammation is fundamental for protecting the organism against infection and injury. However, a failure to control immune response results in chronic inflammation and several associated disorders such as pain and loss of function. Initiation of inflammation is orchestrated by cytokines, among which IL-1β is particularly important. IL-1β is synthesized as an inactive protein that has to be processed by the inflammasome to generate the mature bioactive form. Conventional techniques cannot monitor IL-1β activation with high spatial and temporal resolution. In this study, we present a ratiometric biosensor that allows monitoring IL-1β processing in real time, with a temporal resolution of seconds and with a single-cell spatial resolution. Using this sensor, to our knowledge, we describe for the first time the kinetic of the inflammasome activity in living macrophages.With this new probe, we also demonstrated that the pro-IL-1β processing occurs all over the cytoplasm. Copyright © 2012 by The American Association of Immunologists, Inc.