Staphylococcus aureus (S. aureus) is a major cause of human morbidity and mortality worldwide. Hemolysis caused by S. aureus cytotoxins is important for the acquisition of iron and subsequent bacterial survival during infection. S. aureus can express numerous hemolysins that have been shown to target human erythrocytes. However, the relative importance of each of these for causing hemolysis during pathogenesis in humans is not clear. In this study, we have examined the hemolytic capacity of different methicillin-resistant S. aureus (MRSA) deletion mutants against human erythrocytes in suspension using two separate assays. The first assay measured hemolysis caused by extracellular factors produced by MRSA, while the second measured hemolysis following co-culture of MRSA with human erythrocytes. Results from both assays demonstrated that phenol-soluble modulin-α peptides (PSMα) play a dominant role in causing hemolysis of human erythrocytes, highlighting a prominent target for novel therapeutic strategies designed to limit S. aureus iron acquisition and survival during human disease.