An HPLC-MS/MS method for quantitative determination of the new antituberculosis drug PBTZ169 in blood serum, urine, and feces using perindopril as an internal standard was developed and validated. The method was selective, specific, accurate, and precise. Calibration curves were linear in the ranges 2 – 2,000, 5 – 3,000, and 100 – 10,000 ng/mL for blood serum, urine, and feces, respectively, with correlation coefficients >0.99. The detection limits of PBTZ169 were 1, 2.5, and 50 ng/mL for serum, urine, and feces, respectively. The limits of quantitation were 2 ng/mL in serum; 5, urine; and 100, feces. © 2018, Springer Science+Business Media, LLC, part of Springer Nature.