Muscle stiffness provides a key insight toward clinical assessment for rehabilitation. Regarding the high-cost and technical skill requirement of ultrasound shear wave elastography (SWE) restricting extensive clinical use, MyotonPRO has been proposed as a complementary tool for muscle stiffness measurement. There is a deficiency of studies revealing the use of this tool for measuring muscle stiffness contributing to hand control. The purpose of this study was to assess the capability and effectiveness of MyotonPRO and SWE for hand muscle stiffness characterization. The stiffness of the dominant flexor digitorum superficialis (FDS) muscle of 25 healthy participants (12 males and 13 females) aged 29.60 ± 9.81 years was evaluated while they performed grip tasks. The muscle stiffness of males and females was compared. The correlation between dynamic muscle stiffness given by MyotonPRO and Young’s modulus obtained from SWE was investigated. Statistical analysis indicated a significant difference in the dynamic muscle stiffness between genders in all conditions (p < 0.05), whereas a significant difference in Young’s modulus was found only at the resting state. A moderate correlation was found between dynamic muscle stiffness and Young’s modulus (r ranged from 0.243 to 0.489). Therefore, MyotonPRO can be used to assess the muscle stiffness of the FDS muscle at rest and during muscle contraction.