Gait analysis is a well-established tool for the quantitative assessment of gait disturbances providing functional diagnosis, assessment for treatment planning, and monitoring of disease progress. There is a large volume of literature on the research use of gait analysis, but evidence on its clinical routine use supports a favorable cost-benefit ratio in a limited number of conditions. Initially gait analysis was introduced to clinical practice to improve the management of children with cerebral palsy. However, there is good evidence to extend its use to patients with various upper motor neuron diseases, and to lower limb amputation. Thereby, the methodology for properly conducting and interpreting the exam is of paramount relevance. Appropriateness of gait analysis prescription and reliability of data obtained are required in the clinical environment. This paper provides an overview on guidelines for managing a clinical gait analysis service and on the principal clinical domains of its application: cerebral palsy, stroke, traumatic brain injury and lower limb amputation.