Definitive diagnostics and strict procedures during kidney donor qualification are required. Nowadays, precise and accurate imaging techniques are at hand for every diagnostician. However, many studies have described intraoperative occurrence of horseshoe kidney. Although the harvesting procedure in the case of horseshoe kidney is not technically difficult, graft separation for successful renal transplantation is a challenge. The complex anatomy of malformed organs causes issues during kidney separation. This procedure may lead to damage of the collecting urinary system as well as vascularization damage. Separate graft transplantation is probable when a thin isthmus in a horseshoe kidney is present. Otherwise, poor graft function may occur. We present a technique for horseshoe kidney separation with the use of methylene blue for vascularization determination. The above-mentioned procedure was performed with the methylene blue solution dose injected into a single renal graft artery. Even with the malformed organ's thick isthmus, the exact incision line was identified, exposing vascular perfusion asymmetry and allowing precise renal graft separation.