The present research study focuses on the development of the high‐performance thin‐layer chromatographic method using the design of experiment approach for the simultaneous estimation of Rifampicin and Quercetin. Chromatographic separation was performed on Aluminium plates precoated with silica gel 60 F254 using chloroform:methanol:formic acid:ethyl acetate:benzene (4:1.8:0.3:1.7:1, v/v/v/v/v) as optimized mobile phase. A fractional factorial design was applied for the robustness study and the independent variables selected were mobile phase composition (A), solvent front (B), chamber saturation time (C), and wavelength (D). It was statistically revealed that the volume of ethyl acetate affected the Rf of both drugs resulting in stricter control of ethyl acetate volume compared to the other three variables. Rf for Rifampicin and Quercetin was 0.35 and 0.71 at 347 nm. The linear concentration range was 100–600 ng/band for both drugs. The %recovery ranged between 95.25% and 96.71% for Rifampicin and 95.33%–96.86% for Quercetin. The method was validated by determination of linearity, precision, accuracy, and specificity according to Q2(R1) guidelines, and the %relative standard deviation values were less than 2% for both drugs. A simple, accurate, and reproducible high‐performance thin‐layer chromatographic method was developed, for routine quality control testing of pharmaceutical formulation.