The present study was aimed at developing a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) method for simultaneous determination of curcumin (CRM) and gefitinib (GFT) in bulk, plasma and brain homogenate. hydrochlorothiazide was used as an internal standard (IS). A new simple, rapid, selective, precise and accurate RP-HPLC method has been developed. The separation was achieved by using C-18 column (Qualisil BDS C18, 250 mm x 4.6 mm I.D.) coupled with a guard column of silica, mobile phase consisted of acetonitrile: water with 0.1% formic acid (30:70 v/v). The flow rate was 0.2 ml/min and the drug was detected using PDA detector at the wavelength of 242 nm. The experimental conditions, including the diluting solvent, mobile phase composition, column saturation and flow rate, were optimised to provide high-resolution and reproducible peaks. The method was developed and tested for linearity range of 10-60 μg/mL for bulk analysis and 200-800 ng/mL for plasma and brain homogenate. The developed method was validated as per ICH guidelines, in terms of linearity, application of the proposed method to bulk sample, recovery, precision, repeatability, ruggedness, sensitivity (LOD and LOQ) and robustness and stability study (short and long-term stabilities, freeze/thaw stability, post-preparative). The low value of % RSD showed that the method was precise within the acceptance limit of 2%. The developed method was successfully applied for the analysis of the drug in bulk as well as various marketed formulation and drug in plasma and brain distribution studies.