Clavulanic acid (CA) is an important secondary metabolite commercially produced by cultivation of Streptomyces clavuligerus (Sc). It is a potent inhibitor of bacterial β-lactamases. In this work, a specific and improved high performance liquid chromatography (HPLC) method, using a C-18 reversed phase column, diode array detector and gradient elution for CA quantification in fermentation broths of Sc, was developed and successfully validated. Samples were imidazole-derivatized for the purpose of creating a stable chromophore (clavulanate–imidazole). The calibration curve was linear over a typical range of CA concentration between 0.2 and 400 mg/L. The detection and quantification limits were 0.01 and 0.02 mg/L, respectively. The precision of the method was evaluated for CA spiked into production media and a recovery of 103.8%, on average, was obtained. The clavulanate–imidazole complex was not stable when the samples were not cooled during the analysis. The recovery rate was 39.3% on average. This assay was successfully tested for CA quantification in samples from Sc fermentation, using both, a chemically defined and a complex medium.