Guideline-directed treatment with penicillin or other β-lactam antibiotics is commonly hampered by penicillin allergy labeling. For a study, researchers sought to test a standardized approach to distinguish non-allergic patients from those with real allergic β-lactam hypersensitivity. They used a retrospective de-labeling approach in 800 patients with suspected β-lactam hypersensitivity. Between 2009 and 2019, everyone got a comprehensive allergy work-up that allowed them to exclude definitively or diagnose β-lactam allergy. Negative challenge testing ruled out β-lactam allergy in 595 (74.4%) of the 800 patients studied. About 70 individuals (8.7%) had IgE-mediated anaphylaxis, while 135 had delayed-type hypersensitivity (16.9%). The algorithm properly recommended an alternative antibiotic in 62 (88.6%) anaphylactic cases. Patients with moderate to severe anaphylaxis had higher accuracy (97.7%) than those with a history of favorable responses (73.1%). The system properly identified 122 (90.4%) patients diagnosed with delayed-type hypersensitivity. It allowed de-labeling in 330 (55.5%) of the 595 individuals with the diagnostic exclusion of penicillin hypersensitivity but failed to identify the other 265 (44.5%) as low-risk instances. The algorithm correctly identified penicillin (β-lactam) allergy in 89.8% of instances, and its sensitivity was excellent for moderate to severe anaphylaxis. As part of an antibiotic stewardship program, the results support the introduction of a consistent de-labeling algorithm under close supervision to allow guideline-directed treatment and limit the usage of broad-spectrum antibiotics.