UV–Vis absorption spectroelectrochemistry has been selected as a suitable multiresponse technique to determine the fluoroquinolone ofloxacin in urine samples without any previous pretreatment. Due to the modification of the working electrode surface during the oxidation of this antibiotic, the electrode has to be replaced or polished between measurements. A new spectroelectrochemistry cell has been developed, which allows in an simple way to perform reproducible experiments with different screen-printed electrodes. The new cell has been used to perform analysis in a complex matrix such as urine. Due to the trilinear character of the spectroelectrochemical data, PARAFAC has been used to determine ofloxacin with very good figures of merit, demonstrating the capability of trilinear methods to avoid the influence of some interfering compounds.