In this protocol, we provide step-by-step instructions for dissection and culture of primary murine dorsal root ganglia (DRG), which provide an opportunity to study the functional properties of peripheral sensory neurons in vitro. Further, we describe the analysis of neuropeptide release by ELISA as a possible downstream application. In addition, isolated DRGs can be used directly for immunofluorescence, flow cytometry, RNA sequencing or proteomic approaches, electrophysiology, and calcium imaging. For complete details on the use and execution of this protocol, please refer to Perner et al. (2020).