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Background: Telaprevir is a protease inhibitor used in the treatment of hepatitis C virus infection. Analytical methods for telaprevir should separate the compound from its R-diastereomer VRT-127394, which is 30-fold less active. The objective of this work was to develop liquid chromatography-tandem mass spectrometer (LC-MS/MS) assays for telaprevir both in plasma and in dried blood spot (DBS), capable of stabilizing the equilibrium and chromatographically separating the 2 epimers., Methods: Human plasma was acidified with formic acid and frozen within 1 hour after collection to stabilize the equilibrium between the 2 telaprevir diastereomers ex vivo in plasma. After protein precipitation, the sample was analyzed with LC-MS/MS. For the DBS assay, sampling paper was impregnated with citric acid solution to achieve stabilization of the epimers on the sampling paper. DBS samples were extracted before LC-MS/MS analysis. LC-MS/MS analysis comprised online solid-phase extraction and separation on a C18 column, with the mass spectrometer operating in TurboIonSpray-negative ionization mode and performing multiple reaction monitoring., Results: The assays were linear over the concentration range of 0.1-10 mg/L in plasma and DBS. Accuracies ranged from 97% to 106% in plasma and from 93% to 99% in DBS. Within- and between-day coefficients of variation were <7.9% in plasma and <9.3% in DBS. Human whole blood samples with hematocrit values of 27%-47% gave reproducible quantitation results in the DBS assay, and spot volume did not affect results of the DBS assay either. Acidified plasma with telaprevir was stable for 5 hours at 20°C, and telaprevir on impregnated DBS paper was stable for at least 3 months at 4°C or at 20°C., Conclusions: An assay was developed and validated for the determination of telaprevir in human plasma, separating telaprevir from its R-diastereomer VRT-127394. In addition, a DBS assay was developed, which avoids immediate centrifuging, acidification, and freezing of patient samples to stabilize the equilibrium between the 2 telaprevir diastereomers.