Abstract: Production of proinflammatory cytokines in the central nervous system is a key process in the neuroinflammatory response to trauma, infection, and neurodegenerative diseases (Kumar, 2019). These intercellular signaling molecules play multiple roles in the immune response in the central nervous system including the orchestration of the sickness response to innate immune perturbations in the brain (Dantzer et al., 2008). Brain innate immune cells such as microglia and other macrophages (perivascular, meningeal) are considered a significant source of cytokines (Ransohoff and Cardona, 2010) during neuroinflammatory conditions. Thus, quantification of cytokines in the central nervous system is essential to understanding the neuroimmune mechanisms underpinning neuroinflammatory conditions and to monitor the effects of treatment. However, quantification of brain cytokines has largely been limited to end-point measures of tissue protein levels of cytokines using techniques such as enzyme-linked immunosorbent assay (ELISA), western blot assay, or immunohistochemistry, which fail to discriminate between intracellular and extracellular levels of cytokines. In other words, an experimental change in total tissue levels of cytokines does not necessarily mean that the protein was secreted into the interstitial space within a brain region. Proinflammatory cytokine receptor antagonists as well as germ-line knockouts have been employed to block the behavioral, physiological, and neuroinflammatory response to stress (Goshen and Yirmiya, 2009) and immune challenge (McCusker and Kelley, 2013), which implicates, but does not directly demonstrate cytokine release in the brain. A further limitation of measuring cytokines in whole tissue is that measurements are restricted to a single time point post-mortem. This limitation necessitates using a between-subjects experimental design to conduct time course measurements of cytokines, which introduces error variance due to between-subject variability in biological responses. Notably, inflammatory cytokines such as interleukin (IL)-1β have very short half-lives (Liu et al., 2021). Thus, methods that are limited to measuring single time points post-immune challenge lack the temporal resolution to capture the rapid kinetic changes in inflammatory cytokines. In this Perspective piece, we explore a recent technological advance that allowed us to serially quantify cytokines within the interstitial space of discrete brain regions of freely behaving rodents. This approach not only permits quantification of cytokine release into the extracellular space, but also provides increased spatial and temporal resolution of cytokine release in the brain under neuroinflammatory conditions.