Background: Choosing to perform squats on an unstable surface potentially offers advantages surpassing those of their stable counterpart. Objectives: The purpose of this study was to compare muscle activation during squats on stable and unstable surfaces and investigate the relationship among thigh muscles. Design: Observational study. Methods: Nineteen adults participated in this study. The stable surface consisted of a flat floor. The unstable surface involved the use of an air cushion. An air cushion was positioned beneath each foot of the study participants. Surface electromyography was employed to measure muscle activation. %MVC was calculated by measuring muscle activation during squat execution and manual muscle testing. Results: The comparison of muscle activation during squats between stable and unstable surfaces revealed a significant difference in the rectus femoris and biceps femoris (P<.05). On stable surfaces, positive correlations were observed in the rectus femoris, vastus lateralis, and vastus medialis (P<.05). On unstable surfaces, positive correlations were found between the rectus femoris and vastus medialis (P<.05). Conclusion: This study observed an increase in muscle activation of the rectus femoris and biceps femoris during squats on an unstable support surface compared to a stable support surface.