Biomechanical testing of long bones can be susceptible to errors and uncertainty due to malalignment of specimens with respect to the mechanical axis of the test frame. To solve this problem, we designed a novel, customizable alignment and potting fixture for long bone testing. The fixture consists of three-dimensional-printed components modeled from specimen-specific computed tomography (CT) scans to achieve a predetermined specimen alignment. We demonstrated the functionality of this fixture by comparing benchtop torsional test results to specimen-matched finite element models and found a strong correlation (R2 = 0.95, p < 0.001). Additional computational models were used to estimate the impact of malalignment on mechanical behavior in both torsion and axial compression. Results confirmed that torsion testing is relatively robust to alignment artifacts, with absolute percent errors less than 8% in all malalignment scenarios. In contrast, axial testing was highly sensitive to setup errors, experiencing absolute percent errors up to 50% with off-center malalignment and up to 170% with angular malalignment. This suggests that whenever appropriate, torsion tests should be used preferentially as a summary mechanical measure. When more challenging modes of loading are required, pretest clinical-resolution CT scanning can be effectively used to create potting fixtures that allow for precise preplanned specimen alignment. This may be particularly important for more sensitive biomechanical tests (e.g., axial compressive tests) that may be needed for industrial applications, such as orthopedic implant design.