Background: Squatting is one of the best functional exercises to strengthen the quadriceps muscle in independent gait and activities of daily living. Although the use of a gym ball during squat exercise is the most common way of strengthening the vastus medialis oblique (VMO) muscle, published data on this subject are limited. Objects: The purpose of this study was to compare the sequential muscle activation of the VMO and vastus lateralis (VL) muscles during squat exercise using a gym ball at different knee flexion angles. Methods: Forty healthy adults were randomly divided into experimental (squat exercise using a gym ball) and control (squat exercise alone) groups, in which squats were performed at 45° and 90° knee flexion. Electromyographic (EMG) activity data were collected over 10 seconds under the 2 angles of knee flexion (45° and 90°). Results: There was significant group and time interaction effect for VMO and VL muscle activation at 45° knee flexion. This was similarly demonstrated at 90° knee flexion. No significant group main effect and time main effect for VMO and VL muscle activation were noted at 45° knee flexion, respectively. In contrast, there was significant group main effect and time main effect for VMO and VL muscle activation at 90° knee flexion. These significant differences were demonstrated through two-way analysis of variance over repeated measurements, suggesting that the EMG activity of the VMO muscle during squatting with a gym ball showed remarkable improvement compared to that of the VL muscle. Conclusion: This research suggests that squat exercise using a gym ball may be more beneficial in improving the activity of VMO than of the VL muscle at both 45° and 90° of knee flexion, respectively. We highly recommend squat exercises with a gym ball for selective strengthening of the VMO muscle in knee rehabilitation.