Although combinational antiretroviral therapy has been proven highly effective, it suffers from drug-drug interactions, drug resistance and adverse reactions with long-term use. The introduction of novel drugs in antiretroviral therapy proposes newer treatment options. However, drug safety and their potential interactions after long-term therapy remain largely unexplored. In this study, the induction potential of bictegravir on efflux transporters at the protein level was assessed by quantifying the transporters using an LC-MS/MS-based method. A surrogate peptide approach was used for the simultaneous determination of P-gp, BCRP and MRP1 transporter proteins in rat peripheral blood mononuclear cells. The previously developed Excel-based ProteoExcel