Background Rotator cuff tendinopathy (RCT) is a leading cause of shoulder pain and disability. Management is mainly conservative, but the limited ability of tendons to regenerate is the main cause of unsatisfactory results. So, we conducted our study to compare the efficacy of deep prolotherapy (glucose 25%), platelet-rich plasma (PRP), and betamethasone corticosteroid for treatment of RCT to find the most effective one based on clinical, functional, and radiological assessment. Results Regarding visual analog scale (VAS), it was significantly (p < 0.001) improved after injection among group 1 (prolotherapy group) and group 3 (steroid group) patients, while no significant improvement was noted among group 2 (PRP group) (p = 0.212) patients. The Western Ontario Rotator Cuff (WORC) Index significantly improved among the studied groups (p < 0.001, p = 0.049, and p < 0.001, respectively) after injection. Regarding the range of motion (ROM), a significant improvement (p = 0.029) was achieved in group 1 after injection but no significant improvements were noted among group 2 and 3 patients (p = 0.529 and 0.121, respectively). There was a significant improvement among group 1 and 2 patients (p < 0.001 and p = 0.020, respectively) regarding the grade of tendon lesions but no improvement occurred among group 3 patients (p = 0.470). Conclusion Prolotherapy injections improve shoulder ROM, VAS, WORC index, and rotator cuff tendon healing while PRP injections improve WORC index and tendon healing but steroid injection has no effect on healing. Trial registration PACTR202005610509496. Retrospective registration on May 25, 2020, Pan African Clinical Trial Registry.