Background: The scapulo-thoracic musculatures including serratus anterior (SA), upper trapezius and lower trapezius can provide shoulder stability and functional shoulder movement. Objects: The muscle activities of upper and lower SA were compared during three different scapular protraction exercises in healthy individuals in sitting position. Methods: Twenty-five healthy subjects were participated. Electromyography device was used to measure muscle activity of upper and lower SA and trapezius muscles. Each subject was asked to perform three different scapular protraction exercises (scapular protraction [SP], SP with self-resistance [SPSR], SPSR with hand-exerciser [SPSRH]) in random order. One-way repeated measures analysis of the variance and a Bonferroni post hoc test were used. Results: The muscle activity of lower SA muscle was significantly different among three conditions (SP vs. SPSR vs. SPSRH) (p < 0.01). The lower SA muscle activity was significantly greater during SPSRH compared to SP and SPSR, which required joint stability more than SP and SPSR (p < 0.01). Conclusion: SPSRH exercise can be recommended to facilitate the muscle activity of lower SA. In addition, the intramuscular variation in the upper and lower SA during scapular protraction exercise is required to consider the effective rehabilitation.