Thymoquinone (THY) is a bioactive compound present in the seed powder of Nigella sativa (NS). This research aims to precisely and accurately estimate THY using high-performance liquid chromatography (HPLC) with a Quality by Design (QbD) application. Box-Behnken design (BBD) was employed to optimize the chromatographic conditions for HPLC method development, taking mobile phase flow rate, pH of the buffer, and λmax as independent variables and retention time and tailing factor as the measured responses. The mobile phase composition was methanol: acetonitrile: buffer (2.2 mM ammonium formate) at the ratio of 35:50:15 v/v/v on a Symmetry® C18 (5 μm, 3.9 × 150 mm) column. In isocratic mode, it had a flow rate 0.9 mL min−1 and eluted analyte was detected at 249 nm. Validation parameters followed the International Council for Harmonization (ICH) guidelines for the new HPLC method. The method was linear over the range 6.25–100 µg mL−1 with a coefficient of determination (r2) of 0.9957. The limit of detection (LOD) and limit of quantification (LOQ) were 2.05 and 6.25 µg mL−1, respectively. The %RSD of system suitability for retention time was 1.42% and for the tailing factor it was 0.695%. In addition, the developed method was precise, accurate, and robust according to ICH criteria. The developed HPLC method is simple, accurate, quick, and robust, and it could be used for the routine analysis of THY in different kinds of formulations.