Laboratory gait analysis or three-dimensional gait analysis (3DGA), which uses motion capture, force plates and electromyography (EMG), has allowed a better understanding of the underlying mechanisms of gait deterioration in patients with multiple sclerosis (PwMS). This review will summarize the current knowledge on multiple sclerosis (MS)-related changes in kinematics (angles), kinetics (forces) and electromyographic (muscle activation) patterns and how these measures can be used as markers of disease progression. We will also discuss the potential causes of slower walking in PwMS and the implications for 3DGA. Finally, we will describe new technologies and methods that will increase precision and clinical utilization of 3DGA in PwMS. Overall, 3DGA studies have shown that functionality of the ankle joint is the most affected during walking and that compensatory actions to maintain a functional speed may be insufficient in PwMS. However, altered gait patterns may be a strategy to increase stability as balance is also affected in PwMS.