A robust and simplified high-performance liquid chromatography (HPLC) method was developed for the estimation of Pyrazinamide and its related substance. A systematic approach, one of the parts of QbD (quality by design) was used in suitable analytical method development. The HPLC segregation method was carried out with C-18 Column (3.9x300 mm, I. D. 10 μm), a mobile phase of phosphate buffer: Acetonitrile (pH 3.0) 90:10 v/v, detected at 270 nm. Optimization to this method was done by response surface methodology by applying a three-level Box Behnken design with three center points. © 2019 Deuton-X Ltd. All rights reserved.