The present work aimed to assess the validity and reliability of the 3D motion analyzer (Shimano Dynamics Lab, Sittard, Netherland) during laboratory cycling tests in comparison with the Vicon device (Vicon Motion Systems Ltd. Oxford, UK). Three cyclists were required to complete one laboratory cycling test at three different pedalling cadence and at a constant power output. Kinematic measurements were collected simultaneously from 3D motion analyzer and Vicon devices and performed five times for each pedalling cadence. The two systems showed a high reliability with excellent intraclass correlation coefficients for most kinematic variables. Moreover, this system was considered as valid by considering the error due to the initial markers placement. Experts and scientists should use the Vicon system for the purpose of research whereas the 3D motion analyzer could be used for bike fitting.