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Objective: To evaluate the safety and accuracy of a unilateral three-dimensionally printed animal-specific drill guide (3DASDG) design for unilateral stabilization in the thoracolumbar vertebral column of dogs compared to a bilateral design. Study design: Cadaveric study. Sample population: Fifty-two corridors in one canine cadaver. Methods: Two 3DASDG designs with 2 drilling tubes each were created from T8 to L7 vertebrae. Fifty-two corridors were drilled on the right and the left sides by using unilateral and bilateral designs, respectively. Planned and postoperative trajectories (entry point, exit point, angle) were compared to establish the accuracy. Statistical analysis was used for accuracy comparison between designs. Safety was evaluated by using Zdichavsky classification. Results: Unilateral and bilateral drill guide designs were not different for entry point and angle deviations; however, they were different for the exit point deviations. Two corridors breached outside the vertebra. For all guides, mean entry and exit point deviations were less than 1 and 2 mm, respectively. The maximum entry or exit point deviation in both groups was 4.7 mm. The mean angle deviation was