Importance: Affordable, quantitative methods to screen children for developmental delays are needed. Motor milestones can be an indicator of developmental delay and may be used to track developmental progress. Accelerometry offers a way to gather real-world information about pediatric motor behavior. Objective: To develop a referent cohort of pediatric accelerometry from bilateral upper extremities (UEs) and determine whether movement can accurately distinguish those with and without motor deficits. Design, Setting, and Participants: Children aged 0 to 17 years participated in a prospective cohort from December 8, 2014, to December 29, 2017. Children were recruited from Ranken Jordan Pediatric Bridge Hospital, Maryland Heights, Missouri, and Washington University School of Medicine in St Louis, St Louis, Missouri. Typically developing children were included as a referent cohort if they had no history of motor or neurological deficit; consecutive sampling and matching ensured equal representation of sex and age. Children with diagnosed asymmetric motor deficits were included in the motor impaired cohort. Exposures: Bilateral UE motor activity was measured using wrist-worn accelerometers for a total of 100 hours in 25-hour increments. Main Outcomes and Measures: To characterize bilateral UE motor activity in a referent cohort for the purpose of detecting irregularities in the future, total activity and the use ratio between UEs were used to describe typically developing children. Asymmetric impairment was classified using the mono-arm use index (MAUI) and bilateral-arm use index (BAUI) to quantify the acceleration of unilateral movements. Results: A total of 216 children enrolled, and 185 children were included in analysis. Of these, 156 were typically developing, with mean (SD) age 9.1 (5.1) years and 81 boys (52.0%). There were 29 children in the motor impaired cohort, with mean (SD) age 7.4 (4.4) years and 16 boys (55.2%). The combined MAUI and BAUI (mean [SD], 0.86 [0.005] and use ratio (mean [SD], 0.90 [0.008]) had similar F1 values. The area under the curve was also similar between the combined MAUI and BAUI (mean [SD], 0.98 [0.004]) and the use ratio (mean [SD], 0.98 [0.004]). Conclusions and Relevance: Bilateral UE movement as measured with accelerometry may provide a meaningful metric of real-world motor behavior across childhood. Screening in early childhood remains a challenge; MAUI may provide an effective method for clinicians to measure and visualize real-world motor behavior in children at risk for asymmetrical deficits.