Acromioclavicular joint (ACJ) disruption occurs frequently in athletes engaged in contact sports. However, the current understanding of ACJ biomechanics during muscle-driven functional activities and the influence of different treatment approaches (eg, reconstruction surgery vs nonoperative methods) on ACJ kinematics and stability remains limited. The absence of precise in vivo biomechanical measurement modalities for scapular and clavicular kinematics contributes significantly to this lack of understanding. The purposes of this study were to determine whether dynamic stereo x-ray (DSX) imaging can be used to evaluate the in vivo kinematics of the ACJ and to provide preliminary comparative data on ACJ kinematics, range of motion, and isometric strength of surgically reconstructed or nonoperatively treated ACJ shoulders and their uninjured contralateral shoulders. It was hypothesized that ACJ kinematics could be measured successfully using DSX and that surgically and nonoperatively treated shoulders would show abnormal 3-dimensional (3-D) ACJ kinematics compared with the uninjured contralateral. Controlled laboratory study. In this cross-sectional study, 11 participants who had undergone unilateral ACJ reconstruction surgery and 3 patients who received nonoperative treatment were enrolled. ACJ kinematics were assessed during active forward flexion, scaption, and abduction through high-speed DSX imaging, complemented by 3-D bone models obtained via computed tomography (CT) scans. To gauge kinematic differences, a 1-dimensional statistical parametric mapping method was employed, which compared outcomes in the index limb to those in the uninjured counterpart. In addition, the range of motion and isometric strength at various abduction angles were analyzed, employing a repeated-measures analysis of variance to compare the affected and uninjured sides. Leveraging a combination of DSX imaging and patient-specific CT bone models, ACJ kinematics was measured successfully during movements along anatomic planes. Preliminary findings from this investigation revealed no detectable differences between the surgically reconstructed and uninjured sides in ACJ biomechanics, shoulder range of motion, and isometric strength outcomes. However, on average, the nonoperatively treated shoulders demonstrated increased internal rotation, upward rotation, and posterior tilting of the scapula relative to the clavicle (no statistical analyses were performed due to the small sample size). DSX imaging is a promising tool for evaluating potential in vivo kinematic abnormalities in the ACJ during muscle-driven activities, laying the groundwork for further investigations in both ACJ-reconstructed and nonreconstructed patients. This study furnished essential data for conducting power analyses and designing future studies with an adequate sample size to investigate the impact of different treatment approaches on shoulder girdle mechanics. With its potential for accurately characterizing shoulder girdle kinematics post-ACJ injury, DSX imaging can offer valuable insights for future clinical studies, facilitating informed decisions regarding the short- and long-term impacts of treatment choices on shoulder health and function.