Abstract Two-component mixtures of cefsulodin and clavulanic acid were analysed by a first-derivative spectrophotometric method using a zero-crossing technique of measurement. The relative ease offered by this derivative technique for the quantification of these drugs, with closely overlapping spectral bands, was clearly demonstrated. As the absorption band of clavulanic acid closely overlaps with that of cefsulodin, both direct and derivative spectrophotometric methods have been investigated and evaluated by an exhaustive statistical analysis of the experimental data. The first-derivative spectrophotometric method was found to be more rapid, accurate and reproducible. The procedure does not require any separation step. The calibration graphs were linear in the range 2.0–56.0 μg ml −1 for cefsulodin and 2.0–28.0 μg ml −1 for clavulanic acid. The lower detection limits of cefsulodin and clavulinic acid ( P 0.05 level) were calculated to be 0.16 and 0.24 μg ml −1, respectively. Mixtures of cefsulodin and clavulanic acid in ratios of 1:4–7:2 were satisfactorily resolved. Both components were also determined in physiological solutions used to prepare intravenous infusions of these antibiotics.