Microglia are central nervous system (CNS) resident macrophages. They play a prominent role in virtually all neurodegenerative and traumatic brain injuries. Visualizing microglia using label-free methodologies will allow a better understanding of how microglia participate in CNS disorders in the absence of perturbations from external fluorescent dyes. Fluorescence Lifetime Imaging (FLIM) of NADH is an effective tool for monitoring cell intrinsic metabolic changes, and can imply metabolic state based on free/bound NADH lifetime quantification. Recently, FLIM based NADH lifetime quantification was used to characterize macrophages and other immune cell types. Here, we use a lifetime-based, label-free method to characterize microglia in vitro. We have found that there is a unique and statistically significant difference (p<0.05, n=5) in the NADH lifetime and free/bound NADH levels in microglia compared to other CNS cell types. Activated (i.e. inflammatory) microglia play a particularly important role in CNS diseases compared to quiescent microglia, and to our knowledge, there are no markers that can uniquely identify activated microglia, and distinguish them from related immune cell types. Thus, we have extended our NADH FLIM-based approach to differentiate quiescent microglia from activated microglia and found that there is a statistically significant difference (p<0.05,n=5) in NADH mean lifetime in the activated cells. Identifying microglia in a mixed population of CNS cells, and distinguishing activated from quiescent microglia will pave the way to better understanding their roles in the healthy and diseased brain.