In this study, the liquid chromatographic (LC) parameters were optimized using response surface methodology (RSM) as a novel approach to achieve optimal separation of six vitamers of vitamin D and K during simultaneous estimation. Analytes were separated using an Accucore C18 column (50 x 4.6 mm, 2.6 µm), 0.1% aqueous formic acid (pH = 3.5), and methanol as mobile phase components. The Box-Behnken design (BBD) predicted the best combination of the selected critical quality attributes such as organic solvent composition in the mobile phase (90%), mobile phase flow rate (0.42 mL/min), and column oven temperature (40oC). Multiple regression analysis was used to fit the experimental data from 17 sample runs to a second-order polynomial equation. The adjusted coefficient of determination (R2) for three desired responses were 0.983 (retention time of K3 = R1), 0.988 (resolution between D2 and D3 = R2), and 0.992 (retention time of K2-7 = R3), all with significant probability values (p<0.0001), indicating a high significance for the regression model. Q-ToF/MS detection was interfaced with an electrospray combined ionization source. The optimized detection parameters delivered specific, sensitive, linear, accurate, precise, and robust quantification of all six analytes in the tablet dosage form.