Motor function involves complex physiologic processes and requires the integration of multiple systems, including neuromuscular, musculoskeletal, and cardiopulmonary, and neural motor and sensory-perceptual systems. Motor-functional status is indicative of current physical health status, burden of disease, and long-term health outcomes, and is integrally related to daily functioning and quality of life. Given its importance to overall neurologic health and function, motor function was identified as a key domain for inclusion in the NIH Toolbox for Assessment of Neurological and Behavioral Function (NIH Toolbox). We engaged in a 3-stage developmental process to: 1) identify key subdomains and candidate measures for inclusion in the NIH Toolbox, 2) pretest candidate measures for feasibility across the age span of people aged 3 to 85 years, and 3) validate candidate measures against criterion measures in a sample of healthy individuals aged 3 to 85 years (n = 340). Based on extensive literature review and input from content experts, the 5 subdomains of dexterity, strength, balance, locomotion, and endurance were recommended for inclusion in the NIH Toolbox motor battery. Based on our validation testing, valid and reliable measures that are simultaneously low-cost and portable have been recommended to assess each subdomain, including the 9-hole peg board for dexterity, grip dynamometry for upper-extremity strength, standing balance test, 4-m walk test for gait speed, and a 2-minute walk test for endurance.