AbstractTelaprevir is a potent, selective, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3‐4A serine protease. it is used for the treatment of HCV infection in combination with peginterferon alfa and ribavirin. In the present work, the E–Z isomerization process of telaprevir in solution was revealed by online HPLC–DAD (diode array detector)–MS, variable‐temperature and variable‐gradient experiments. The molecular geometry information of the two isomers was established by molecular mechanics calculations, and good correlation between the two isomers' UV–vis spectra and their molecular geometry information was also discovered. In addition, it was revealed by molecular docking that the two isomers have different affinities to HCV NS3•4A protease, and the Z isomer, the minor form of telaprevir in solution, is the more effective inhibitor of HCV NS3•4A protease. The investigation can provide more structure information about telaprevir in solution and in the binding process of HCV NS3•4A protease.