Background and Purpose: Elamipretide is a mitochondrial-targeting agent being developed for the treatment of mitochondrial dysfunction-associated diseases. While prior studies have shown that subcutaneous elamipretide is generally safe/well tolerated, injection site reactions (ISRs) were reported in most subjects. We evaluated the efficacy of interventions to mitigate ISRs, identify underlying ISR mechanisms, and evaluate the pharmacokinetic and safety profile of subcutaneous elamipretide. Experimental Approach: Subcutaneous elamipretide 60 mg was administered to healthy subjects (N=10) on six separate occasions with/without potential ISR interventions (mometasone furoate, ice application, tacrolimus ointment, doxepin cream, and oral diphenhydramine). ISR clinical/self-assessments, blood samples, and safety data were collected at predetermined intervals. Preclinical studies investigated mast cell-specific receptor MRGPRX2 mediation of ISRs. Key Results: Mometasone significantly reduced the incidence of induration/swelling and pruritus. Diphenhydramine significantly decreased the incidence of induration; 50% reported somnolence. Ice application significantly reduced the incidence of pain, although it reduced elamipretide’s maximum plasma concentration and area-under-the-curve from time 0-6hrs versus elamipretide alone. Preclinical data suggest that SQ-elamipretide induced ISRs by activating MRGPRX2 in humans and its ortholog Mrgprb2 in mice. Conclusion and Implications: Elamipretide activated MRGPRX2 and Mrgprb2 receptors, resulting in activation of mast cells and inflammation in mouse models, suggesting that targeting mast-cell activation may ameliorate elamipretide ISRs. Topical mometasone prior to subcutaneous elamipretide demonstrated significant reductions in ISR signs and symptoms and did not cause significant changes in elamipretide plasma exposure or additional adverse events. Therefore, mometasone prior to subcutaneous injection of elamipretide warrants further investigation in clinical studies for alleviating ISRs.