An analytical method was developed for the determination of bosentan and its three main metabolites in various matrices and species with focus on robustness. The drug assay involved protein precipitation, followed by liquid-liquid extraction and column switching in combination with narrow bore HPLC-MS-MS. Deuterated analogues of the analytes were used as internal standards. The sample preparation procedure was optimised with respect to minimise the suppression effects from different matrices. The drug and its metabolites could be analysed in plasma, serum, bile, and liver samples from man, dog, and rat with a run cycle time of 10 min. The method used always calibration samples made up in human plasma, whereas quality control samples were prepared in human plasma as well as in the identical matrix as the unknown samples. Calibration graphs for the drug and for the metabolites were linear in the range from 1 or 2 to 2000 or 10,000 ng/ml using a sample volume of 0.25 ml. Mean inter-assay precision and accuracy were 3.0% and 98.4%, respectively. Two additional methods were derived from the main method for the analysis of plasma samples only with focus on reduced manual effort and instrumental run cycle time. The modified methods showed a mean inter-assay precision and accuracy of 5.0% and 99.9% for the method using column-switching, and 3.5% and 98.8% for the method using off-line SPE, respectively. All methods proved to be robust, sensitive, and selective during the analysis of several thousand samples.