A mass spectrometry method has been developed using the Quality by Design (QbD) principle. Direct analysis in real time mass spectrometry (DART-MS) was adopted to analyze a pharmaceutical preparation. A fishbone diagram for DART-MS and the Plackett-Burman design were utilized to evaluate the impact of a number of factors on the method performance. Multivariate regression and Pareto ranking analysis indicated that the temperature, determined distance, and sampler speed were statistically significant (P < 0.05). Furthermore, the Box-Behnken design combined with response surface analysis was then employed to study the relationships between these three factors and the quality of the DART-MS analysis. The analytical design space of DART-MS was thus constructed and its robustness was validated. In this presented approach, method performance was mathematically described as a composite desirability function of the critical quality attributes (CQAs). Two terms of method validation, including analytical repeatability and method robustness, were carried out at an operating work point. Finally, the validated method was successfully applied to the pharmaceutical quality assurance in different manufacturing batches. These results revealed that the QbD concept was practical in DART-MS method development. Meanwhile, the determined quality was controlled by the analytical design space. This presented strategy provided a tutorial to the development of a robust QbD-compliant mass spectrometry method for industrial quality control.