The posterior retroperitoneoscopic adrenal access represents a challenge in orientation and working space creation. The aim of this experimental acute study was to evaluate the impact of computer-assisted quantitative fluorescence imaging on adrenal gland identification and assessment of intraoperative remnant perfusion for adrenal resection in the posterior retroperitoneoscopic approach.Six pigs underwent simultaneous (n = 5) or sequential (n = 1) bilateral posterior retroperitoneoscopic adrenalectomy (n = 12). Fluorescence imaging was obtained via intravenous administration of 3 mL of Indocyanine Green (ICG) and by switching the camera systems to near-infrared mode (D-LIGHT P, KARL STORZ; Germany). Fluorescence-based visualization of adrenal glands before vascular division (n = 4), after the main vascular pedicle ligation (negative control, n = 1) or after adrenal resection (n = 7), was followed by completion adrenalectomy. The fluorescence signal intensity dynamics were recorded and analyzed using proprietary software. For each pixel, the slope of fluorescence signal intensity evolution over time was translated into a color-coded perfusion cartography, which was superimposed onto real-time images obtained with the corresponding left and right camera systems. Quantitative fluorescence signal analysis in the regions of interest (ROIs) served to assess adrenal remnant perfusion in divided adrenal glands.In the retroperitoneum, the vascular anatomy was illuminated in fluorescence imaging first. The adrenal glands were promptly highlighted after primary intravenous ICG administration (n = 9) or showed a fluorescence signal intensity increase upon reinjection (n = 3). Quantitative fluorescence analysis showed a statistically significant difference between perfused and ischemic segments in divided glands (p = 0.0156).Fluorescence imaging provides real-time guidance during minimally invasive adrenal surgery. Prior to dissection, it allows to easily discriminate the adrenal gland from surrounding retroperitoneal structures. After adrenal gland division, ICG injection associated with a computer-assisted quantitative analysis helps to distinguish between well-perfused and ischemic segments. Further studies are underway to establish the correlation between remnant perfusion and viability.