Background: The first dorsal interosseous (FDI) and opponens pollicis (OP) muscles are the main stabilizers of the trapeziometacarpal joint. Optimal exercise selection based on surface electromyography (sEMG) activity may help to optimize rehabilitation programs in hand therapy. Purpose: To compare the sEMG activity of FDI and OP during seven typical rehabilitation exercises. Study Design: Cross-sectional. Methods: Twelve healthy participants (age: 25.92 ± 5.79, female/male: 6/6) were included. The sEMG activity of the FDI and OP was measured during seven therapeutic exercises; thumb-to-little fingertip pinch, tri-digit tip pinch, intrinsic plus fist with ball, key pinch, four- and five-finger separation with elastic resistance, and thumb-index tip pinch. Root mean square was used and adjusted to the maximum voluntary contraction (MVC) of each muscle. Friedman's test with Dunn's post-hoc was used to compare amplitude signals between exercises. Results: Intrinsic plus fist with ball showed the highest electromyographic amplitude for FDI and OP. Post-hoc analysis revealed a statistical difference between intrinsic plus fist with ball (45.67% of MVC) and four-finger separation (8.70% MVC, p < 0.0001) for the OP. While for FDI, post-hoc analysis showed statistical differences between intrinsic plus fist with ball (49.70% MVC) and four hand exercises (thumb-to-little fingertip pinch = 12.13% MVC, p < 0.0001; tri-digit tip pinch = 13.21% MVC, p < 0.0001; four-finger separation = 18.51% MVC, p = 0.004; thumb-index tip pinch = 17.66% MVC, p = 0.0028). Discussion/Conclusion: Intrinsic plus fist with ball exercise showed the highest sEMG activation. These findings could guide hand therapists in the optimal selection of exercises, taking into account the level of muscle activity in routine rehabilitation exercises.