This study presents the development of a new RP-UPLC analytical method for the quality control of a pharmaceutical product, according to Quality by Design (QbD) guidelines. QbD is a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding. Risk assessment and Design of Exeriments (DoE) represent fundamental tools of Quality by design approach. QbD leads to the establishment of the Design Space (DS), namely the multidimensional space of combinations of variables that have been demonstrated to provide assurance of Quality. DS can be defined only by using multivariate techniques such as DoE. A scouting system was set-up to test different organic phases, organic ramps, starting and ending conditions to approach to the conditions leading to good selectivity and fast time of analysis. At the end of the scouting step, the QbD study could start with the screening study of the effect of several chromatographic parameters on different chromatographic responses. the Quality Risk Assessment of the RP-UPLC method was performed using Ishikawa diagram. preliminary screening studies of the chromatographic critical factors, identified in vials type, antigens concentration, injection volume, ramp time, organic concentration, column type and temperature were performed to investigate the knowledge space of the method and to approach to the operative conditions leading to ensure satisfactory sensibility and selectivity.Two asymmetric screening matrices were set-up to study the critical factors by DoE. The graphic study of effects made it possible to fix the optimum value for some factors and to identify a newexperimental domain for the factors to be further investigated. Finally, Response Surface Methodology was used to identify the DS, as the multidimensional combination of variables where the requirements for the critical quality attributes of the method were satisfied with a selected probability.