BACKGROUND: Tuberculosis is a worldwide health concern and isoniazid is the most used and considered one of the most effective drugs for its treatment. The "quality" concept must be incorporated into the final pharmaceutical product, according to the quality by design (QbD) definition. Therefore, the determination of analytical test conditions is extremely important and the design of experiments (DoE) becomes a very useful tool. OBJECTIVE: This paper used the concept of QbD to assist the development of analytical conditions for isoniazid and its respective prodrug, applying HPLC. METHOD: HPLC analytical methodologies were developed for isoniazid and its succinylated derivative. The experimental design was carried out using three analytical parameters at three levels. Four chromatographic responses were studied. The impact of analytical parameters on chromatographic responses was assessed using a Pareto chart. Regression models were obtained using multiple regression analysis. DoE analysis was conducted using the Minitab® program and the experiments were performed sequentially, with varying factors. RESULTS: We identify three main risk parameters: mobile phase (high), flow rate (moderate), and pH of buffer (moderate). The ratio of mobile phase buffer (X2) and mobile phase pH (X3) had a major influence on the peak resolutions (Y3). The capacity factors for iso-suc (Y1) and isoniazid (Y2) peaks should be within 3-9 and 4-10, respectively. The peak resolutions between iso-suc and isoniazid (Y3) should be above two. CONCLUSIONS: We designed 27 experiments, obtaining 1.0 mL/min flow rate, 95% buffer in the mobile phase, and pH 7.0 as the optimal analytical conditions. HIGHLIGHTS: Analytical Quality by Design was used as an important tool to determine the best analytical test conditions for isoniazid and its respective prodrug - succinylated isoniazid.