The quantification of the concentration of antibiotics is important to improve their efficacy and for dose correction in cases where the PK/PD of the drug is altered. This is very clear in critically ill patients or when certain clearance procedures such as dialysis or hemofiltration are used. However, there are no techniques that allow such quantification on a routine basis. For this reason, Wong and co-workers developed a fluorescent biosensor based on a mutant beta-lactamase (PenPC) to qualitatively sense the presence of beta-lactam antibiotics in agricultural and food samples. In this paper, a method will be established that allows the determination of these compounds in a routine manner through the use of the PenPC biosensor.