Abstract: A flow injection technique with fluorimetric detection was described for the sequential determination of 6-methoxy-7-hydroxycoumarin (scopoletine) and 7-hydroxycoumarin (umbelliferone) without prior separation. A single-line flow injection system equipped with a double-injection valve was employed to allow the simultaneous injection of sample (40 µL) and phosphate buffer (100 µL) into an aqueous 50% ethanol carrier stream (1 ml/min). The buffer-sample-buffer plug was propelled through the mixing coil (0.5 m x 0.5 mm i.d.) to the detector where the fluorescence at 418 nm was measured (excitation at 350 nm). Buffer solutions of pH 6 and 11 were injected sequentially. A calculation method based on differential fluorimetry was used to determine 1 µM-scopoletine in the presence of a 9-fold excess of umbelliferone or 1 µM-umbelliferone in the presence of a 23-fold excess of scopoletine. The analysis of synthetic binary mixtures containing 24 µM-coumarins yielded recoveries of 96-115%. The technique can be applied to the analysis of binary mixtures of other fluorescent compounds which exhibit pH-dependent excitation spectra.