Probiotics offer numerous health benefits, yet safety concerns persist regarding certain strains capable of producing cytolysins—pore-forming toxins that compromise cellular integrity and contribute to tissue damage. Enterococcus faecalis, a commonly studied strain, exhibits both probiotic potential and pathogenic traits, particularly when cytolysin production is involved. Accurate assessment of cytolysin production is essential to ensure the safety of probiotic strains in therapeutic and commercial applications. Traditional in vitro methods such as hemolysis assays, Polymerase Chain Reaction (PCR), Enzyme-Linked Immunosorbent Assay (ELISA), and proteomic techniques provide valuable insights, but mass spectrometry (MS) has emerged as a powerful tool for detecting and characterizing cytolysins with high sensitivity and specificity. This chapter explores the application of MS in evaluating cytolysin production, highlighting its advantages in identifying toxin profiles, quantifying expression levels, and understanding the molecular mechanisms underlying cytolysin activity. A comprehensive approach utilizing MS can enhance the risk assessment of probiotic strains, ultimately contributing to safer microbiological applications in healthcare and industry.