A simple, accurate, rapid, and sensitive spectrofluorimetric method for the determination of ofloxacin in urine was developed by means of first derivative matrix isopotential synchronous fluorescence spectrometry (MISF). The calibration curve was found to be linear in the concentration range 40-320 ng/mL. The method allows the determination of compound in samples with unknown background fluorescence without the need for tedious pre-separation. Synchronous scans are performed along a trajectory that connects points of identical intensity in a three-dimensional fluorescence spectrum. The unknown analytical signal of the urine is suppressed from the MISF spectrum by calculating its first derivative at ?<sub>ex</sub> = 319.2 and ?<sub>em</sub> = 465 nm. In order to ensure maximum sensitivity and adequate selectivity, the experimental variables affecting fluorescence intensity were studied in the ofloxacin band centered at ?<sub>ex</sub> = 333 and ?<sub>em</sub> = 460 nm. As result, the determination was performed in a water medium at pH 7.2, adjusted by using sodium dihydrogen phosphate as a buffer solution. Calibration graphs were subjected to a comprehensive statistical analysis. The detection limit according to Long and Winefordner was 8.4 ng/mL, and the detection limit proposed by Clayton was 13.9 ng/mL.

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