Thoracoscopy is replacing open lung biopsies because it is less invasive, usually the technique is done using three portals and intracorporeal suture technique. This study described the feasibility and efficacy of a novel pre tied loop ligature and to propose a thoracoscopic access strategy with two portals to perform lung biopsy in patients under 5 kg. Ten rabbits were positioned in dorsal recumbency. Total thoracoscopic lung biopsy was performed using a combined transdiaphragmatic approach and a right intercostal approach. A pre tied loop ligature was placed to perform a caudal lung lobe biopsy. Insufflation of the thoracic cavity was not performed. The total surgery time was 41.4 ± 14.5 min. The procedure was carried out free of complications that prevented slippage or tightening the knot or that made it come loose after the lung biopsy; there was no serious complication during the surgical procedure. The samples obtained averaged 1x0.64x0.45 cm (Length, Width, Depth) and were considered satisfactory according to the histopathologic evaluation. Thorax radiographs taken before and after the surgeries were compared and showed no pneumothorax or hemothorax. Necropsy confirmed no knot failure occurred at the biopsy site. The use of the novel pre tied loop ligature is a safe and effective technique, avoiding problems with the limited size of the thoracic cavity in small patients.