Toxin-producing gram-positive bacteria are responsible for emerging and life-threatening infections in humans worldwide. Both rapid toxin detection and adapted therapy are essential to limit the morbidity due to such toxins, especially staphylococcal Panton-Valentine leukocidin (PVL). Here we describe the use of a mass spectrometry profile generated by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) followed by ClinProTools 2.0 software analysis to find a reproducible model able to identify PVL in Staphylococcus aureus strains. Eighty-one S. aureus strains were used and tested for the presence of PVL, toxic shock syndrome toxin (TSST-1) and mecA genes. The peak at 4448 mass-to-charge ratio (m/z) was the most relevant peak to differentiate between PVL-producing and non-PVL-producing S. aureus. A model using only this peak had an overall recognition capability of 100% and an overall cross-validation of 77.07%. Prospective evaluation of the model allowed two cases of PVL-producing strains to be detected within a few minutes during the time of care and before polymerase chain reaction (PCR) results. Our study represents a proof of concept for the use of such rapid technology as a point-of-care method to identify potential lethal toxin quickly. We believe that such a rapid method will be timely to help change the therapeutic strategy and could be used in the future for other pathogens and infectious diseases.