Introduction: Accurate measurement of physical behavior in adults with lower limb absence is essential to report true patterns of physical behavior and the effectiveness of interventions. The effect of placing accelerometers on prostheses may also affect the reliability and validity. Purpose: To assess reliability and criterion-related validity of the activPAL for measuring incidental and purposeful stepping, and reclining and stepping time in adults with unilateral lower limb absence. Methods: 15 adults with unilateral lower limb absence completed simulated lifestyle activities in a laboratory setting that were retrospectively scored via video analysis. Objective data were obtained simultaneously from two activPAL monitors placed on the sound and prosthetic side. Data were analyzed using one-way intraclass correlation coefficients (ICC), paired t-tests and Cohen’s d. Results: Reliability (prosthetic side vs. sound side) was poor for incidental steps (ICC = .05, d = 0.48) but acceptable for all other measures (ICC = .77–.88; d = .00–.18). Mean activPAL measures, although highly related to the criterion, underestimated, on average, stepping and time-related variables. Differences were large for all stepping variables (d = .38–.96). Conclusions: The activPAL is a reliable measurement tool in adults with lower limb absence when used in a laboratory setting. Placement of the monitor on the sound side limb is recommended for testing. The activPAL shows evidence of relative validity, but not absolute validity. Further evaluation is needed to assess whether similar evidence is found in free-living activity and sedentary contexts.