This study presents the development of gradient reverse phase high performance liquid chroma-tographic method for the determination of Doxorubicin hydrochloride and its five impurities following Quality by Design (QbD) approach and also to identify the conditions where adequate separation quality in minimal analysis duration could be achieved within a robust region that guarantees the stability of method performance. The rela-tionship between critical processparameters and critical quality attributes is created applying Design of Experi-ments methodology. The defined mathematical models and central composite design are used to evaluate the risk of uncertainty in models prediction and concerns in adjusting the process parameters and to identify the design space. Moreover, Box-Behnkan design is applied for experimental robustness testing and method is partially validated to verify the adequacy of selected optimal conditions. The analytical column Waters X-bridge C18 (250 mm x 4.6 mm, 5μm particle size); mobile phase A consisted of buffer (100 mM Sodium dodecyl sulphate and 22mM of Ortho-phosphoric acid , pH adjusted to 2.5 with 1 M sodium hydroxide solution),acetonitrile and methanol (60:30:10) v/v/v; mobile phase B consisted of Buffer, acetonitrile and methanol (30:60:10),column temperature 40ºC, mobile phase flow rate 1 mL min-1, wavelength of detection 254 nm.