Despite advancements in pedicle screw design and surgical techniques, the standard steps for inserting pedicle screws still need to follow a set of fixed procedures. The first step, known as establishing a pilot hole, also referred to as a pre-drilled hole, is crucial for ensuring screw insertion accuracy. In different surgical approaches, such as minimally invasive or traditional surgery, the method of creating pilot holes varies, resulting in different pilot hole profiles, including variations in size and shape. The aim of this study is to evaluate the biomechanical properties of different pilot hole profiles corresponding to various surgical approaches. Commercially available synthetic L4 vertebrae with a density of 0.16g/cc were utilized as substitutes for human bone. Four different pilot hole profiles were created using a 3.0mm cylindrical bone biopsy needle, 3.6mm cylindrical drill, 3.2-5.0mm conical drill, and 3.2-5.0mm conical curette for simulating various minimally invasive and traditional spinal surgeries. Two frequently employed screw shapes, namely, cylindrical and conical, were selected. Following specimen preparation, screw pullout tests were performed using a material test machine, and statistical analysis was applied to compare the mean maximal pullout strength of each configuration. Conical and cylindrical screws in these four pilot hole configurations showed similar trends, with the mean maximal pullout strength ranking from high to low as follows: 3.0mm cylindrical biopsy needle, 3.6mm cylindrical drill bit, 3.2-5.0mm conical curette, and 3.2-5.0mm conical drill bit. Conical screws generally exhibited a greater mean maximal pullout strength than cylindrical screws in three of the four different pilot hole configurations. In the groups with conical pilot holes, created with a 3.2-5.0mm drill bit and 3.2-5.0mm curette, both conical screws exhibited a greater mean maximal pullout strength than did cylindrical screws. The strength of this study lies in its comprehensive comparison of the impact of various pilot hole profiles commonly used in clinical procedures on screw fixation stability, a topic rarely reported in the literature. Our results demonstrated that pilot holes created for minimally invasive surgery using image-guided techniques exhibit superior pullout strength compared to those utilized in traditional surgery. Therefore, we recommend prioritizing minimally invasive surgery when screw implantation is anticipated to be difficult or there is a specific need for stronger screw fixation. When opting for traditional surgery, image-guided methods may help establish smaller pilot holes and increase screw fixation strength.