Fever is one of the most common symptoms of illness in infants and represents a clinical challenge due to the potential for serious bacterial infection. As delayed treatment for these infections has been correlated with increased morbidity and mortality, broad-spectrum β-lactam antibiotics are often prescribed while waiting for microbiological lab results (1-3 days). However, the spread of antibiotic resistance via the β-lactamase enzyme, which can destroy β-lactam antibiotics, has confounded this paradigm; empiric antibiotic regimens are increasingly unable to cover all potential bacterial pathogens, leaving some infants effectively untreated until the pathogen is characterized. This can lead to lifelong sequela or death. Here, we introduce a fluorescent, microfluidic assay that can characterize β-lactamase derived antibiotic susceptibility in 20 min with a sensitivity suitable for direct human specimens. The protocol is extensible, and the antibiotic spectrum investigated can be feasibly adapted for the pathogens of regional relevance. This new assay fills an important need by providing the clinician with hitherto unavailable point of care information for treatment guidance in an inexpensive and simple diagnostic format.