In recent years, several studies have demonstrated the crucial role played by the sympathetic spleen innervation in regulating immune cell function involving fighting pathogens or tissue injury. These findings have sparked interest across different research fields with a common goal of understanding and manipulating splenic sympathetic activity to modulate immune function and inflammation. However, the anatomical identification of spleen-projecting neurons in rodents presents a considerable challenge, given the multi-compartmentalized location of their cellular components. This article addresses this challenge by providing a detailed anatomical dissection guide of the mouse celiac ganglion and splenic nerve, harboring the cell body and projecting axons, respectively. By combining antero- and retrograde neuronal tracing, immunofluorescence, 3D reconstruction, and viral tracing techniques, we validate the connectivity between the celiac ganglion and the spleen and provide insights into their microanatomy. Importantly, we demonstrate the feasibility of viral transduction in these neurons. Additionally, we identified nerve-associated macrophages (NAMs) within the splenic nerve and demonstrated their responsiveness to inflammatory stimuli. Our findings offer a comprehensive anatomical framework for studying spleen-projecting neurons, paving the way for future investigations into their role in immune regulation and inflammation, as well as their manipulation using advanced neurobiological tools.