Scapular dyskinesia, glenohumeral internal rotation deficit, upper posterior labral anterior tears, and rotator cuff injuries are common in athletes who play “overhead” sports due to their repetitive excessive movements. The aims of this study are to propose a new protocol with kinematic analysis coupled with sEMG and to objectively analyze the effect of a specific prevention exercise protocol. Thirty-two subjects (age: 22 ± 4 years, height: 183 ± 3.2 cm, BMI: 23 ± 0.96 kg/m2), including sixteen healthy subjects (Group A) and sixteen male water polo athletes (Group B), underwent a three-dimensional motion analysis based on optoelectronic and sEMG systems. A functional evaluation was performed on Group A and Group B to assess the reliability of the operator-dependent tasks and collect a series of normative data, before starting the prevention protocol (T0) and after 8 weeks (T1). The athletes performed a specific exercise protocol to prevent shoulder injuries. In Group B, the movements of abduction (T0: 111° ± 24°; T1: 140° ± 13°) and extension (T0: 72°± 10°; T1: 84° ± 2.8°) of the glenohumeral joint and the scapulothoracic joint (T0: 33° ± 8.36°; T1: 40.5° ± 10.6°) significantly improved. A significant reduction (in %) in the maximum voluntary contraction (MCV) at T1 of the upper trapezius, teres minor, and pectoralis major was observed. This protocol provides objective data in a simple and reliable way for the functional assessment of the shoulder in water polo players during the sport season.