The present article utilized analytical quality by design (AQbD) methodology to optimize chromatographic conditions for the routine analysis of Cholecalciferol (CHL). Taguchi orthogonal array design and Box-Behnken design were employed to screen and optimize critical method parameters for augmenting the method performance. The optimal chromatographic separation was attained on Eurosphere® 100-5, C8 (250 × 4.6 mm i.d., 5 Î¼m) column in an isocratic elution mode using methanol: acetonitrile (50:50, % v/v) as mobile phase at a flow rate of 1.0 mL /min and Photodiode array detection at 265 nm. The optimized chromatographic method was successfully validated as per ICH Q2 (R1) guidelines. The method was found to be linear (r2 = 0.9993) in the range of 20–100 IU/mL. LOD and LOQ were found to be 10 and 20 IU/mL. The precision, robustness and ruggedness values were within the acceptance limits (RSD
Cholecalciferol, Analytical Quality by design, Taguchi orthogonal array design, Box-Behnken design, Method validation, Forced degradation studies.