β-LEAF assays can be used to determine activity of multiple antibiotics simultaneously. β-LEAF assays were set up with multiple antibiotics (cefazolin, cefoxitin and cefepime) in selected S. aureus isolates. Antibiotic activity was assessed in positive control strain #1, negative control strain #2 and four S. aureus clinical isolates that showed substantial β-lactamase production (#6, #18, #19, #20). The different bacterial strains were incubated with β-LEAF alone and β-LEAF and cefazolin/cefoxitin/cefepime respectively. Fluorescence was monitored over 60 min. The y-axis represents cleavage rate of β-LEAF (measured as fluorescence change rate – milliRFU/min) normalized by bacterial O.D. (optical density) at 600 nm. Results are presented as the average of three independent experiments (each experiment contained samples in triplicates) and error bars represent the standard error.