Neural stem cells (NSCs) in the adult brain are primarily quiescent but can activate and enter the cell cycle to produce newborn neurons. NSC quiescence can be regulated by disease, injury, and age, however our understanding of NSC quiescence is limited by technical limitations imposed by the bias of markers used to isolate each population of NSCs and the lack of live-cell labeling strategies. Fluorescence lifetime imaging (FLIM) of autofluorescent metabolic cofactors has previously been used in other cell types to study shifts in cell states driven by metabolic remodeling that change the optical properties of these endogenous fluorophores. Here we asked whether autofluorescence could be used to discriminate NSC activation state. We found that quiescent NSCs (qNSCs) and activated NSCs (aNSCs) each have unique autofluorescence intensity and fluorescence lifetime profiles. Additionally, qNSCs specifically display an enrichment of a specific autofluorescent signal localizing to lysosomes that is highly predictive of cell state. These signals can be used as a graded marker of NSC quiescence to predict cell behavior and track the dynamics of quiescence exit at single cell resolution in vitro and in vivo . Through coupling autofluorescence imaging with single-cell RNA sequencing in vitro and in vivo , we provide a high-resolution resource revealing transcriptional features linked to rapid NSC activation and deep quiescence. Taken together, we describe a single-cell resolution, non-destructive, live-cell, label-free strategy for measuring NSC activation state in vitro and in vivo and use this tool to expand our understanding of adult neurogenesis.