A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed to determine bosentan (BOS) and glimepiride (GPD) in human plasma simultaneously. Chromatographic separation was carried out on an Acquity UPLC BEH C18 column and mass spectrometric analysis was performed using a QTrap5500 mass spectrometer coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 552.0 → 202.1 and m/z 491.2 → 125.9 were used to quantify BOS and GPD, respectively. This assay method has been fully validated in terms of selectivity, linearity, recovery and matrix effect, accuracy, precision and stability. The linearity of this method was found to be within the concentration range of 5–1000 ng/mL for BOS, and 2.5–500 ng/mL for GPD in human plasma. Only 1.5 min was needed for an analytical run. This assay was used to support a clinical study where multiple oral doses were administered to healthy Chinese subjects to investigate the pharmacokinetics of BOS and GPD.