Objective: Gait provides a sensitive measurement for signs of aging and neurodegenerative conditions. Measurement of gait is transitioning from the laboratory environment to the clinic with the use of inertial measurement units, providing a simple and cost-effective assessment tool. However, such assessments first need validation against reference systems. The aim of this study was to validate the APDM Mobility Lab (ML) system (version 2) against a pressure sensor walkway in younger adults (n = 18), older adults (n = 18) and people with mild Parkinson's disease (n = 21) in the laboratory. Approach: Participants completed a two-minute walk over a pressure sensor walkway whilst wearing three sensors (strapped to the lumbar spine and both feet). Comparison of output from the systems was then performed. Main results: Overall, we identified that ML provided good to excellent agreement (ICC > 0.75) for gait velocity, stride length, stride length SD, cadence, stride time and stride time SD. Measures of double support time, single support time and swing time had moderate to poor agreement (ICC 0.213–0.725), particularly for younger adults and PD. Significance: Overall, Mobility Lab provides a valid system for gait data collection for clinical and research application.