Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors arising from adrenal and extra-adrenal chromaffin cells that secrete excessive amounts of catecholamines into the bloodstream, causing life-threatening cardiovascular complications. While catecholamine overproduction by tumor burden is well established, emerging evidence indicates that dysregulation of the exocytotic machinery itself contributes to pathological secretion. MicroRNAs (miRNAs) act as post-transcriptional inhibitors controlling molecular components of exocytosis involved in trafficking, docking, and fusion of dense-core secretory vesicles containing catecholamines. Yet their specific role in PPGLs remains incompletely understood. We employ a multi-faceted approach combining primary cultures from surgically resected human tumors, advanced single-cell imaging, and comprehensive immunophenotyping. To date, six independent human tumors have been successfully dissociated, maintained in primary culture, and transfected with miRNAs, demonstrating the experimental feasibility of the project. We focus on miR-34a-5p, identified in a previous study as a miRNA that regulates exocytosis. We analyze exocytosis using Total Internal Reflection Fluorescence (TIRF) microscopy with NPY-pHluorin as a fluorescent reporter of exocytosis, enabling quantitative analysis of single vesicle fusion events at the plasma membrane under basal and stimulated (acetylcholine) conditions. Analysis of TIRF images is performed using IVEA, an AI-based software that enables detection and quantification of exocytosis events. We address culture heterogeneity through immunocytochemistry and immunohistochemistry with neuroendocrine and stromal markers, enabling identification of chromaffin tumor cells. This integrated platform establishes a framework for investigating miRNA-regulated exocytosis in human PPGL chromaffin cells and identifying therapeutic targets in hypersecretory neuroendocrine tumors.