The quality by design strategy was applied for the simultaneous chromatographic quantifcation of a binary mixture of bromhexine HCl and its pharmacologically active metabolite ambroxol HCl in dosage forms as well as in human plasma. In this study, fve independent parameters were screened by fractional factorial design to specify the critical ones. The optimal conditions were determined by a response surface methodology using a central composite design. Response surface methodology enabled the best separation in minimal run time, as well as, prediction of separation and retention parameters with minimum error. Separation and quantitation were carried out on BDS Hypersil C8 (250×4.6 mm, 5 μm) RP-column at 1.1 mL min−1 fow rate, 25 mM of KH2PO4 (pH 3.5) in aqueous mobile phase, 65% MeOH, 210 nm wavelength of detection and 10 µL injection volume. After optimization of the chromatographic parameters, validation of the method was achieved according to ICH guidelines. Linearity ranged from 0.195 to 100 µg mL−1 ambroxol HCl, and 0.391–100 µg mL−1 bromhexine HCl, with R2 values of 0.9998 and 0.9999 and limits of detection of 0.098 and 0.195 µg mL−1, respectively. Recovery results ranged from 98.06 to 100.18 and 97.88 to 100.68 for bromhexine HCl and ambroxol HCl, respectively, with RSD less than 1.80.