Objective To test the feasibility of robotic anatrophic nephrolithotomy (RANL) using near-infrared fluorescence (NIRF) image-guidance for treating staghorn stones, in an in vivo stone surgery model. Methods We developed a novel technique of RANL in a preclinical setting following guidelines on safe surgical innovation from the Idea, Development, Exploration, Assessment, Long-term monitoring (IDEAL) collaborative. We performed 2 RANL procedures on 2 live Yorkshire porcine females (IDEAL stage 0 study). The robot was docked in the flank position and a mini-GelPOINT was placed periumbilically as an assistant port. A model staghorn “stone” was created in vivo by injecting low-viscosity DenMat precision material into the renal pelvis. NIRF image-guidance, following clamping of the posterior renal artery, was used to determine if an anatrophic plane could be identified. One procedure was assessed under cold ischemia, with ice-slush injected onto the renal surface via the mini-GelPOINT. Results Both porcine subjects underwent RANL successfully. Replica staghorn models could be created reliably (mean size 5.1 cm; solidification time 2-3 minutes). NIRF image-guidance afforded clear vascular demarcation for precise scoring of an anatrophic plane in both kidneys. The staghorn models were removed in toto through the anatrophic incision in both subjects. Mean blood loss was 160 cc. Mean console and ischemia times were 114 minutes and 34.5 minutes, respectively; ice-slush hypothermia led to a renal surface temperature of 15.4°C. Conclusion In this IDEAL stage 0 preclinical study, we demonstrated that NIRF image-guidance is able to accurately identify the renal avascular plane, thus permitting an anatrophic approach for robotic excision of staghorn stones.