The β-lactam core is a key structure responsible for inducing both IgE-mediated acute-onset hypersensitivity and T-cell-mediated delayed-onset hypersensitivity with penicillins in humans. There is essentially no clinically significant immunologic cross-reactivity noted between the β-lactam cores of penicillins and cephalosporins based on challenge studies in humans. The side-chains appear to be more important in inducing IgE-mediated acute-onset hypersensitivity and T-cell delayed-onset hypersensitivity with cephalosporins in humans. Despite these clinical findings, the U. S. Food and Drug Administration (FDA) still requires the level of β-lactam-related antibiotic residues to be controlled at very low levels in manufacturing facilities. Ceftolozane is Merck & Co., Inc., Kenilworth, NJ, USA's (MSD's) 5th generation broad spectrum cephalosporin antibiotic against gram-negative bacteria. In searching for the optimal decontamination method of ceftolozane, most methods were found to be very slow in opening the β-lactam ring in ceftolozane. Moreover, most of the previously reported decontamination methods applied analytical methods that only monitored the disappearance of the parent molecule as the endpoint of degradation. In this way, many of the β-lactam-containing degradation products could be overlooked. In order to develop an efficient decontamination solution for ceftolozane, a sensitive ultra high performance liquid chromatography-high resolution-electrospray ionization-tandem mass spectrometry (UHPLC-HRMS/MS) method was first developed to ensure the detection of the β-lactam ring in all degradation products. Through online UHPLC-UV-HRMS monitoring, 2.5 N KOH in 50% aqueous MeOH or 50% aqueous EtOH was identified as the best condition to fully degrade the β-lactam ring in ceftolozane. This decontamination could be done within 15 min, even at 100 mg/mL concentration, and thus enable a quick turnaround time for equipment cleaning in the β-lactam manufacturing facility. This method was also successfully applied to 12 other commercially available β-lactam antibiotics.