This study validated the feasibility of inertial sensors in estimating lower limb joint kinematics during stair ambulation in healthy older adults and stroke survivors. Three dimensional motion data were collected using an inertial sensor-based system from 9 persons with stroke and 9 healthy older adults as they ascended and descended a staircase at a self-selected pace. The measured joint angles were compared with a laboratory-based motion capture system by computing differences in range of motion (RoM), grand mean error, standard deviation, and coefficients of multiple correlations. For stroke survivors, differences in RoM measurements between these two systems were determined to be 3.3 ± 8.1°, while the highest correlations were found in the estimation of sagittal plane joint angles after offset correction. Results suggest that the inertial sensor system is suitable for estimating major joint angles in healthy older adults as well as the RoM for stroke survivors. New calibration procedures are necessary for applying the technology to a stroke population.