This study aims to develop and evaluate a cycling-specific marker protocol that minimises the number of markers while accounting for the unique biomechanics of cycling. Although movements in the frontal and transverse planes during cycling are limited, they are clinically relevant due to their association with overuse injuries. Existing gait-based marker protocols often fail to consider cycling-specific factors such as posture, range of motion, marker occlusion, and muscle-induced artifacts. The proposed protocol (PP) uses 15 physical and 8 virtual markers. In the absence of a gold standard for 3D pedalling kinematics, the PP was evaluated by comparing it with established gait analysis protocols. The protocol demonstrated high correlation in gait (CCC > 0.98 for hip and knee in the sagittal plane), low intra-subject variability (CV < 15% for hip, knee, and ankle), and high repeatability. During pedalling, position, velocity, and acceleration were measured in all three spatial directions. Notably, angular velocity and linear acceleration showed significant components outside the sagittal plane, particularly for angular velocity. These findings highlight the importance of considering 3D motion when estimating forces, joint moments, and joint-specific powers in cycling biomechanics.