The human endogenous cannabinoid system (ECS) regulates key physiological processes and alterations in its signaling pathways, and endocannabinoid levels are associated with diseases such as neurological and neuropsychiatric conditions, cancer, pain and inflammation, obesity, and metabolic and different immune related disorders. Immune system cells express the G-protein coupled cannabinoid receptor 1 (CB1), but its functional role has not been fully understood, likely due to the lack of appropriate tools. The availability of novel tools to investigate the role of CB1in immune regulation might contribute to identify CB1as a potential novel therapeutic target or biomarker for many diseases. Herein, we report the development and validation of the first fluorescent small molecule probe to directly visualize and quantify CB1in blood and tonsil immune cells by flow cytometry and confocal microscopy. We coupled the cannabinoid agonist HU210 to the fluorescent tag Alexa Fluor 488, generating a fluorescent probe with high affinity for CB1and selectivity over CB2. We validate HU210-Alexa488 for the rapid, simultaneous, and reproducible identification of CB1in human monocytes, T cells, and B cells by multiplexed flow cytometry. This probe is also suitable for the direct visualization of CB1in tonsil tissues, allowing the in vivoidentification of tonsil CB1-expressing T and B cells. This study provides the first fluorescent chemical tool to investigate CB1expression and function in human blood and tonsil immune cells, which might well pave the way to unravel essential features of CB1in different immune and ECS-related diseases.