Design of Experiment (DoE) is an effective methodology for studying the relationship between variables and their dependent responses. In this study, DoE was employed for method development and optimization. A binary combination of safinamide mesylate (SAF) and its synthetic precursor p-hydroxybenzaldehyde (PHB) were analyzed by RP-HPLC. Method development focused on evaluating the effect of three independent factors; % EtOH, pH, and flow rate; on performance and efficiency of separation. A rotatable central composite design (CCD) was applied to determine crucial variables and optimize key performance parameters. Significant interest has been shown in green analytical chemistry as a means to reduce the use of hazardous organic solvents without degrading chromatographic performance. A green separation and quantification were performed using a Waters VDSpher PUR 100 C18-E column (5 µm, 250 × 4.6 mm) at a flow rate of 1 mL/min, utilizing a binary combination of mobile phase A: B (60:40, v/v). Phase A of the mobile phase consisted of 0.02 M ammonium formate and 0.02 M ammonium acetate, with the pH adjusted to 5.5 using formic acid. EtOH was utilized as mobile phase B, and 20 µL was employed as the injection volume. The technique was validated in accordance with ICH criteria. SAF had a linear range of 4–100 µg/mL, while PHB had a linear range of 0.2–10.0 µg/mL. Both analytes had R2 values of 0.9999, with detection limits of 0.95 and 0.06 µg/mL, respectively. The designed method could be applicable for regular QC analysis of SAF and its pharmaceutical product. GAPI and AGREE algorithms, which use 12 principles of green analytical chemistry, were used to evaluate the method greenness.