Abstract A specific high-performance liquid chromatographic assay was developed for a simultaneously qualitative and quantitative determination of clivorine, a hepatotoxic otonecine-type pyrrolizidine alkaloid, and its four putative hepatotoxicity-related metabolites, namely dehydroretronecine, 7-glutathionyldehydroretronecine, 7,9-diglutathionyldehydroretronecine, and clivoric acid, generated in rat microsomal incubation. This simultaneous determination was conducted by a direct analysis of aliquots of the supernatant of incubates using a specific two-column set-up. Impurities in the supernatant were firstly eluted out from the first PRP-1 guard column (50×4.1 mm) during an initial 5 min washing period with isocratic elution by mobile phase A (0.2% formic acid at pH 3.4 adjusted by ammonia). Subsequently, the guard column was then connected to the second PRP-1 analytical column (250×4.6 mm) and analytes were separated by a gradient elution with mobile phases A and B (acetonitrile). The assay provided good reproducibility and accuracy for all analytes tested with less than 12% of overall intra- and inter-day variations and higher than 87% of overall accuracy. This developed method was successfully applied to determine the intact clivorine and its four metabolites generated in rat microsomal incubation.