An automatic system was developed to determine ofloxacin in biological fluids and pharmaceutical formulations. Drug detection was carried out by a potentiometric membrane sensor based on [bis(trifluoromethyl)phenyl]borate as molecular-recognition material. The tubular shaped detector system was solidly attached to the manifold, creating a high-throughput stable setup (50 samples per hour) appropriate for routine antibiotic assessment. Under the optimized flow conditions, the sensor displayed a mean detection limit of 1 × 10(-5) M, a linear response over the concentrations of 2 × 10(-5) to 5 × 10(-3) M (slope of 57.4 mV decade(-1)) and a wide working pH range (2.1 - 6.6). The procedure was successfully applied to ofloxacin analysis in pharmaceuticals (relative deviation lower than 6%) and biological fluids at levels usually found after drug administration of clinical doses (recoveries between 91 and 106%). No significant interference from common excipients found in commercial formulations and inorganic ions usually present in biological fluids was noticed.