Frailty is a clinical syndrome associated with the aging process and adverse outcomes. The purpose of this short report was to initiate the development of a Frailty Index in 27- to 28-month-old C57BL/6 mice that matched the clinical criteria used in humans (weakness, slow walking speed, low activity level, poor endurance). The selected criteria included grip strength, walking speed, physical activity, and endurance. The criteria in mice were evaluated by the inverted-cling grip test, rotarod test, voluntary wheel running, and derived endurance scores. Each criterion had a designated cutoff point (1.5 SD below the cohort mean) to identify the mice with the lowest performance. If a mouse presented with three of the criteria scores below the cutoff points, it was identified as frail. Mild frailty was designated if two criteria were below the cutoff points. In this mouse cohort, one mouse was identified as frail and one was mildly frail. This prevalence of 9% frailty is consistent with the prevalence of frailty in humans at the same survival age. Collectively, our selected criterion, cutoff point, and Frailty Index provide a potential standardized definition for frailty in mice that is consistent with the operational definition of frailty in humans.