Strength training serves as an effective strategy to prevent and treat various lower limb disorders such as osteoarthritis, muscular weakness and stiffness, imbalance, and injuries. Dysfunctions in the gluteal muscles can lead to pain and functional issues in the ankle, hip, knee, and lumbar spine. Strengthening these muscles is strongly recommended for therapeutic and preventive purposes. Squats are the most well-known and useful exercises. The main objective of this study was to examine the activity of gluteal muscles and the main lower limb muscles during the squat. Motion capture techniques were used to collect kinematics and ground reaction forces for 10 heathy subjects performing five repetitive unloaded squat exercises. For each recorded motion, a musculoskeletal model of the lower limbs using Opensim was used to estimate the muscle activation of the gluteal and lower limb muscles. Results showed that among the gluteal muscles, the Gluteus maximus had the highest activation (23.35% ± 8.79), followed by the Gluteus Medius (17.51% ± 8.74) and Gluteus Minimus (15.53% ± 9.36). However, the lower limb muscles, including Rectus Femoris, Biceps Femoris, Gastrocnemius Medialis, and Tibialis Anterior, showed even higher activations, ranging from 34.43% to 54.58%. This confirms that squats, as basic exercises, contribute to gluteal muscle strengthening, but they may not be considered the most effective exercise for this purpose.