Sarcopenia, an age-related skeletal muscle condition characterized by a progressive decline in muscle mass and function, is linked to increased vulnerability, a higher likelihood of falls, and higher mortality rates in older individuals. A comprehensive understanding of the intricate mechanisms driving skeletal muscle aging is of great significance in both scientific and clinical fields. Consequently, myotube models that facilitate studying regulatory mechanisms underlying skeletal muscle aging are important tools required to advance intervention strategies against skeletal muscle aging and associated disorders. Here, we provide a detailed protocol to generate human pluripotent stem cells-derived myotubes and describe their applications in aging studies, as well as a troubleshooting for potential problems. Overall, this protocol serves as a valuable methodological reference for exploring the role and mechanism of genes involved in skeletal muscle aging.