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Owing to know the complexity in reverse phase high performance liquid chromatography (RP-HPLC) method optimization; i.e., the selection of optimum combination of input variables for achieving the desired separation, a mathematical relationship between the experimental variables with the responses is to be established by response surface methodology. The paper aims to describe the use of the Box–Behnken experimental design to identify the significant variables effects influencing the separation of metaxalone (MTX) and its two base hydrolytic impurities (DP-1 and DP-2) in a stability indicating RP-HPLC method. Influence variables such as column temperature, buffer pH, and buffer concentration, each at three levels were screened for the desirability of responses like theoretical plates, resolution (MTX-DP-1), capacity factor of MTX, tailing factor at 10% (MTX), and analysis time (Rtof DP-2). A statistical program (Design Expert 8.0) was used to calculate and optimize the five responses simultaneously by means of a multiple response optimization algorithm. The optimum conditions are established including the robustness of the method using a Box–Behnken design, as part of the validation exercise. 10 mM phosphate buffer with pH 4.7 at 31°C column temperature was found to be the optimized condition to produce desirable responses. System suitability limits were defined based on the results of the robustness test. The RP-HPLC method was further validated with respect to linearity, range, precision, specificity, accuracy, limit of detection, and limit of quantification, according to the current regulatory requirements. The optimized method gives rapid and efficient separation with complete resolution between the three peaks, and represents an improvement over the existing reported methods. [ABSTRACT FROM AUTHOR]