The misuse of antibiotics in health care has led to increasing levels of drug resistant infections (DRI's) occurring in the general population. Most technologies developed for the detection of DRI's typically focus on phenotyping or genotyping bacterial resistance rather than on the underlying cause and spread of DRI's; namely the misuse of antibiotics. An aptameric based assay has been developed for the monitoring of ampicillin in urine samples, for use in determining optimal antibiotic dosage and monitoring patient compliance with treatment. The fluorescently labelled aptamers were shown to perform optimally at pH 7, ideal for buffered clinical urine samples, with limits of detection as low as 20.6 nM, allowing for determination of ampicillin in urine in the clinically relevant range of concentrations (100 nM to 100 µM). As the assay requires incubation for only 1 h with a small sample volume, 50 to 150 µL, the test would fit within current healthcare pathways, simplifying the adoption of the technology.