Conspectus Signaling lipids, such as the endocannabinoids, play an important role in the brain. They regulate synaptic transmission and control various neurophysiological processes, including pain sensation, appetite, memory formation, stress, and anxiety. Unlike classical neurotransmitters, lipid messengers are produced on demand and degraded by metabolic enzymes to control their lifespan and signaling actions. Chemical biology approaches have become one of the main driving forces to study and unravel the physiological role of lipid messengers in the brain. Here, we review how the development and use of chemical probes has allowed one to study endocannabinoid signaling by (i) inhibiting the biosynthetic and metabolic enzymes; (ii) visualizing the activity of these enzymes; and (iii) controlling the release and transport of the endocannabinoids. Activity-based probes were instrumental to guide the discovery of highly selective and in vivo active inhibitors of the biosynthetic (DAGL, NAPE-PLD) and metabolic (MAGL, FAAH) enzymes of endocannabinoids. These inhibitors allowed one to study the role of these enzymes in animal models of disease. For instance, the DAGL–MAGL axis was shown to control neuroinflammation and the NAPE-PLD–FAAH axis to regulate emotional behavior. Activity-based protein profiling and chemical proteomics were essential to guide the drug discovery and development of compounds targeting MAGL and FAAH, such as ABX-1431 (Lu AG06466) and PF-04457845, respectively. These experimental drugs are now in clinical trials for multiple indications, including multiple sclerosis and post-traumatic stress disorders. Activity-based probes have also been used to visualize the activity of these lipid metabolizing enzymes with high spatial resolution in brain slices, thereby showing the cell type-specific activity of these lipid metabolizing enzymes. The transport, release, and uptake of signaling lipids themselves cannot, however, be captured by activity-based probes in a spatiotemporal controlled manner. Therefore, bio-orthogonal lipids equipped with photoreactive, photoswitchable groups or photocages have been developed. These chemical probes were employed to investigate the protein interaction partners of the endocannabinoids, such as putative membrane transporters, as well as to study the functional cellular responses within milliseconds upon irradiation. Finally, genetically encoded sensors have recently been developed to monitor the real-time release of endocannabinoids with high spatiotemporal resolution in cultured neurons, acute brain slices, and in vivo mouse models. It is anticipated that the combination of chemical probes, highly selective inhibitors, and sensors with advanced (super resolution) imaging modalities, such as PharmacoSTORM and correlative light-electron microscopy, will uncover the fundamental basis of lipid signaling at nanoscale resolution in the brain. Furthermore, chemical biology approaches enable the translation of these fundamental discoveries into clinical solutions for brain dis...