Purpose <p>Determining lumbar spine 3D kinematics requires the reconstruction of medical images. Current imaging modalities, such as Computed Tomography (CT) scans and Magnetic Resonance Imaging (MRI), have limitations, including ionizing radiation and high costs. Musculoskeletal ultrasound (MSU) presents a promising alternative; however, its application in 3D kinematic analysis has not yet been reported. The aim of this study is to assess the reliability and validity of MSU-based 3D reconstruction for determining intervertebral kinematics.</p> Methods <p>Seven lumbar spines were dissected, and technical markers (TMs) were attached to the vertebrae. MSU and CT images were captured, and 3D reconstructed. Discrete motions were applied to the specimens, and the TMs were digitized. The motions were visualized on the 3D reconstructions using a visualization technique. Anatomical landmarks were identified, and reference frames were established. Intervertebral range of motion (ROM) was measured using Euler angles. Reliability was assessed through repeated measurements, and validity was evaluated by comparing the ROM results between the MSU and CT scans.</p> Results <p>The intra-operator reliability showed good (ICC = 0.89 ± 0.01) and excellent (ICC = 0.91 ± 0.01) reliability for the MSU and CT scan, respectively, with slight differences in mean detectable changes (3.23% ± 1.52% for MSU, 2.65% ± 0.75% for CT). Regarding validity no significant mean difference was observed (−&#xa0;0.23° ± 0.20°, p = 0.57) between the mean ROMs, and good agreement was found, with a mean difference of 0.22° ± 0.07° and most data points falling within limits of agreement.</p> Conclusion <p>This study confirms the accuracy and precision of 3D MSU in measuring lumbar spine range of motion, validating its use for 3D kinematic analysis and paves the way for in-vivo analysis.</p>