Neurons in the centromedial amygdala (CeM) are activated during fear memory formation. We designed a new viral vector, AAV9-pfos-hM4Di:mCherry:PEST expressing Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) under a c-fos promoter, enabling the specific manipulation of activated neurons. We use this viral vector to temporarily inactivate the neurons involved during cued-fear learning in the CeM and to uncover their molecular profile.Male and female wildtype C57BL/6J mice are injected with the viral vector in the CeM, exposing one group to the shocks paired with the tones (fear learning), and another to unpaired shocks and tones. Clozapine-N-Oxide (CNO) or a vehicle is injected intraperitoneally 30 minutes after fear learning, and immunohistochemistry (IHC) is used to study cFos expression. In another cohort of animals, 24 hours after fear learning, we also study fear extinction and deltaFosB a marker of repeated neuronal activity by IHC. In a different experiment, afferent neurons to these distinct neuronal subpopulations are studied by injecting the viral vector with a retrograde tracer. To discover the molecular profile of neurons activated during cued-fear learning, another cohort of animals is used for spatial proteomics with whole-slide imaging, laser microdissection, and ultrasensitive liquid chromatography-mass spectrometry.In summary, we posit that leveraging these innovative tools to identify distinct neuronal populations involved in fear learning can significantly enhance our comprehension of previously inaccessible molecular mechanisms underlying memory formation.