Background: Gait assessment is a complex task involving locomotion and balance control across all body segments, requiring a global analysis in the event of motor disorders. Among these are spinal disorders, where an understanding of spinal kinematics during walking is important to improve treatment decisions and outcomes. The technique of stereophotogrammetric motion analysis is currently the gold standard in this context. A new integrated protocol for whole-body kinematic gait analysis is proposed in this study, which takes into account the movements of the spine. Methods: A new protocol with 30 passive markers was developed to analyze gait. Of these markers, 22 implemented the Davis protocol for gait measurement, while the other 8 were placed onto the spine to record spinal movements. The protocol’s accuracy was assessed through comparisons of the constructive angles of a manikin replicating the human body and the angles measured with the optoelectronic system. An assessment of intra- and inter-operator repeatability and protocol usability was carried out by recruiting and applying the protocol in a population composed of ten subjects (mean age 17.36—SD 10.12) without any history of spine pathology. Results: The protocol was validated successfully. The validation accuracy was more than satisfactory: the measured RMSE was 1.2 ± 1° for the data collected with the optoelectronic system with respect to the manikin. The intra-operator repeatability was also good in the sagittal and frontal planes (average ICC > 0.867), and the inter-operator repeatability was moderate or good in all planes (average ICC > 0.77). The usability score obtained using the System Usability Scale was satisfactory (mean 74.75, SD 5.88). Conclusions: This study proposes a new protocol to assess total body kinematics, including the spine in its three main segments, during gait. The successful validation of this protocol in terms of reliability and usability allows for its subsequent clinical application.