Lifestyle heavily influences intervertebral disc (IVD) loads, but measuring in vivo loads requires invasive methods, and the ability to apply these loads in vitro is limited. In vivo load data from instrumented vertebral body replacements is limited to patients that have had spinal fusion surgery, potentially resulting in different kinematics and loading patterns compared to a healthy population. Therefore, this study aimed to develop a pipeline for the non-invasive estimation of in vivo IVD loading, and the application of these loads in vitro. A full-body Opensim model was developed by adapting and combining two existing models. Kinetic data from healthy participants performing activities of daily living were used as inputs for simulations using static optimisation. After evaluating simulation results using in vivo data, the estimated six-axis physiological loads were applied to bovine tail specimens. The pipeline was then used to compare the kinematics resulting from the physiological load profiles (flexion, lateral bending, axial rotation) with a simplified pure moment protocol commonly used for in vitro studies. Comparing kinematics revealed that the in vitro physiological load protocol followed the same trends as the in silico and in vivo data. Furthermore, the physiological loads resulted in substantially different kinematics when compared to pure moment testing, particularly in flexion. Therefore, the use of the presented pipeline to estimate the complex loads of daily activities in different populations, and the application of those loads in vitro provides a novel capability to deepen our knowledge of spine biomechanics, IVD mechanobiology, and improve pre-clinical test methods.