The use of DNA aptamers in biosensors for the quantification of pharmaceuticals in the clinics would help to overcome the limitations of antibody-based detection for small molecules. The interest for such systems is proven by the ever-increasing number of aptamer-based solutions for analytics proposed in the literature as proof-of-concept demonstrators. Despite such diversity, these platforms often lack a comparative assessment of their performances against the current standard of practice in the clinics when using real samples. We employed an aptamer against tobramycin discovered in our laboratory to quantify through surface plasmon resonance the concentration of the antibiotic in clinical samples obtained from patients treated with tobramycin and undergoing therapeutic drug monitoring. We then compared the performances of our detection strategy against the current standard of practice. Our results show how, using adequate calibration and matrix complexity reduction, DNA aptamer-based direct assays can assess clinically relevant concentrations of small molecules in patient serum and with good correlation to current standards used in the clinics.